Advanced Light Microscopy

Our Leica SP8 laser scanning confocal microscopes both feature:

• Inverted platforms

• Pulsed white light lasers for flexible excitation

• Fully automated operational procedures for z-stacks and tiling.

• Differential interference contrast (DIC) imaging

• Environmental control via a Tokai Hit stage-top incubator for live-cell imaging at physiological conditions (5% CO₂ and 37°C)

• Time-gated fluorescence signal collection and single photon counting for HyDs

Leica SP8 FALCON

The Leica SP8 FALCON (Room C1733) is an inverted laser scanning confocal microscope with fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy (FCS) capabilities.

• Software module: LIGHTNING for image enhancement

• Software module: Navigator for overview scans and subsequent acquisitions

Leica SP8 STED 3X

The Leica SP8 STED 3X (Room C1730) is an inverted confocal/STED microscope which achieves super-resolution through the process of 2D/3D stimulated emission depletion (STED).

• Three depletion lasers (592nm, 660nm, 775nm)

• FRET/FRAP software modules

• AFC (hardware autofocus)

• Resonant scanner

Miltenyi Biotec UltraMicroscope II

The UltraMicroscope II (C1733B) allows for fast 3D imaging of tissues and organs with compatibility for many clearing protocols.

Four laser lines: 488nm, 561nm, 640nm, and 785nm

The shape and number of light sheets can be tweaked to match experimental goals.

Imaris Workstation

The Imaris Workstation (located in C1732A) is equipped with a 24-core CPU, 512GB of RAM, an NVIDIA RTX 4090 GPU, and 8TB of NVMe storage.

• Installed commercial software include Imaris and Huygens

• Open-source software include: Fiji, ilastik, Qupath, and Napari.

• Available for independent use with a low hourly rate

• Training/assisted sessions are also available

Commercially licensed software

Huygens Professional

Deconvolve laser scanning confocal and STED datasets using Huygens.

Imaris for Cell Biologists

Segment, track, analyze, and visualize using Imaris.

New users should request a free consultation through email.

While independent usage on the analysis workstations does not require training, a quick consultation is still required before room access is given.

• Scheduling and billing is handled through iLab

• While trained users may book microscopes at any time, timely support from ALM staff is only guaranteed during typical working hours (9am-5pm, Monday-Friday on non-holidays).

• If you cannot make a scheduled session, please cancel as soon as possible to allow for someone else to have a chance to use it.

• If you fail to start your session through the iLab kiosk (and someone books your time), you must yield your time to them.

• As a last resort, bookings may be cancelled for maintenance or repairs.

• Penn State Health credentials are used for logging into the microscope workstations.

• Please reach out with any biosafety concerns concerning new live-cell experiments.

• Please immediately report any problems with the microscopes via email along with relevant pictures or error messages.

• No food is allowed in BSL2 spaces.

• No sitting or leaning on anti-vibration tables.

• Use computers only for iLab, microscope operation, and image processing.

• Unauthorized entry into the facility is prohibited.

• Service contracts do not cover damage from negligence. PIs may be held responsible.

Where to save microscopy files?

Save your microscopy data to the specified local hard drives with the following folder structure: $CurrentYear\$Lab\$LabMemberName\$Experiment

How long will my files stay on the microscope computers?

To ensure sufficient storage, ALM reserves the right to remove data located outside of the designated storage locations or data already synced to the ALM network drive.

How long will my files stay on the ALM network drive?

The ALM network drive is not intended for long-term data storage. Data from the current and previous year will be kept. It is your responsibility to fulfill NIH data requirements, so please back up the files to your lab's network drive or another suitable location.

Data will automatically sync to ALM's network drive every night at

\\main.vast.pennstatehealth.net\files\cores\InstrumentData\ALM

While connected to the PSH network, map to the above drive following the steps for your operating system.

• Instructions for Windows

• Instructions for Mac

Note: You can also try mapping directly to your scope-specific folder.

If you need to access your data immediately after acquisition, copy the data from local storage to your lab's network drive.

Do not use flash drives or external drives.

Shutdown

1. Open an internet browser and sign into iLab.

2. End your kiosk session to avoid extra time charges

3. Check the schedule on iLab to see if someone will be using the microscope soon.

4. Follow the scope-specific shut-down procedures.

Clean-up

Leave no trace.

If immersion objectives were used, carefully clean immersion oil from the oil objectives using lens paper and/or cotton-tipped applicators using Sparkle and/or 70% EtOH. Note: Never allow a used/dirty region of lens paper (or cotton-tipped applicator) to contact the glass front lens.

1. Start by dabbing the metal portion of the objectives with fresh lens paper to remove as much oil as possible from the sides.

2. Finish cleaning the outer metal rim using either lens paper or cotton-tipped applicators with 70% EtOH, making sure never to contact the glass front lens.

3. Dab at the glass front lens with clean lens paper until no more oil is removed.

4. Add Sparkle to a cotton-tipped applicator and gently clean the oil immersion objective by rotating the cotton-tipped applicator.

5. Inspect and confirm that objectives are clean.

Biosafety guidelines

ALM operates as a Biosafety Level 2 laboratory.

• Admission is restricted to authorized (trained) people only unless with ALM staff.

• All human, primate, biohazardous, or genetically modified cells must be assigned appropriate BSL levels for research use by the Institutional Biosafety Committee (IBC) before coming to the Advanced Light Microscopy core. Read the full Institutional Biosafety policy.

• All samples for imaging must be approved in advance by ALM. This is covered by obtaining Biosafety approval.

• All procedures must be performed carefully to minimize the creation of aerosols.

• All liquid and solid waste must be removed as biohazardous material by the user.

• Wear gloves when handling biohazardous samples on the microscope.

• Hands must be washed after handling biohazardous materials and before leaving.

• Neither syringes nor hypodermic needles should be used in the facility without the express consent of ALM. Consent must be obtained each time syringes or hypodermic needles are used--consent today does not imply future consent.

• No eating, drinking, or smoking in the work area.

• No mouth pipetting.

Laser safety guidelines

• Access to the laboratory will be limited to people necessary to the running of experiments when any laser is operating.

• Only qualified personnel shall operate or adjust any laser.

• In an emergency (e.g., fire, water leaks), please act calm, and ensure your safety.

• If possible, shut down and cover equipment before exiting the building.

• Phone the campus emergency line at 717-531-8888, and then notify ALM.