Confocal Microscopy

The Leica TCS SP8 Confocal Microscope:

The SP8 is an inverted laser powered single-point True Confocal Scanning system (TCS) allowing the user to produce publication quality high resolution images of fluorescently labeled histologically fixed tissues and cells, as well as cultured live cells.

Some of the TCS SP8 specifications:

True Confocal Scanning system (TCS) switchable between non-resonant and resonant scanning.  The resonant scanner allows the user to achieve a higher fluorescence quantum yield while reducing photobleaching.  Samples can be scanned at up to 84 frames per second in standard mode or up to 290 frames per second in resonant scanning mode.  Maximum scan resolution is 8192 x 8192 pixels (64 Mpixels) in standard scanning mode, or up to 1024 x 1024 pixels (1 Mpixel) in resonant scanning mode. 

TCS allows optional field rotation up to 200° using a König-Rotator.

The system’s Adaptive Focus Control (AFC) maintains the user’s specified focal plane over time, which improves stability in long-term experiments.

Highly efficient spectral separation is achieved via Leica’s patented unique Pellin-Broca prism design allowing 95% transparency at all wavelengths and polarization orientations.

Leica LIAchroics are low incident angle dichroic beam splitters and are custom designed by Leica Microsystems in-house.  The LIAchroic approach allows a steeper spectral cutoff and higher spectral transmission than other dichroic beam splitter designs resulting in stunning high contrast images.

The patented filter free spectral Leica SP detector allows up to five individually regulated channels.

Spectral detection range includes the visible and infrared range from 400 nm to 800 nm.

The system has three PMT (Photo Multiplier Tube) detectors and two HyD (Hybrid) detectors allowing highly efficient illumination at minimum laser intensity, thus protecting the sample from photobleaching, and one transmitted light brightfield detector.

Long-term or overnight data acquisition of live cell cultures is made possible through the addition of a state-of-the-art live cell incubation chamber provided by OKO labs, Inc.  This chamber maintains proper CO2 levels, proper temperature, and proper humidity levels allowing time extended experimental conditions.

Our SP8 has the following objectives:

• 10x/0.40 CS HC PL APO (dry)
• 20x/0.75 CS2 HC PL APO (dry)
• 40x/1.30 CS2 HC PL APO (oil)
• 63x/1.40 CS2 HC PL APO (oil)
• 100x/1.40 CS2 HC PL APO (oil)

DIC (Differential Interference Contrast) is available with the 20, 40, 63, and 100x objectives for enhanced contrast photos.

The system has a high resolution and highly accurate motorized scanning stage.

Four solid state lasers:

• 405 nm
• 488nm
• 552 nm
• 638 nm

Premium high powered HP Z4G4 CUDA workstation computer:

• Loaded with Windows 10-Professional (64 bit) operating system
• Intel 5122 Xeon 4C 3.6 GHz processor
• NVIDIA Quadro P6000 high performance GPU
• 6 TB SATA hard disc drive
• 30 inch high-brilliance LCD flat screen, true color, 2560 x 1600 pixel monitor
• Leica’s latest LAS-X software package

The core also provides two state-of-the-art stand-alone computers loaded with the full version of Leica LAS X software for users who wish to view and analyze their data at their leisure.

Training:

Training will be provided for all users by the confocal technician (see contact information).  All users must first be trained by the technician before being allowed to use the confocal without assistance.

Reservations:

For all confocal users, simply email or call and discuss your needs with the confocal technician.  We ask that you try to limit your usage of the microscope to half-day increments, especially during days when others are interested in using the scope as well.  

Usage fees:

For all ETSU users and other educational entities:

• $25.00 / hour assisted use
• $25.00 / hour unassisted use

For all other outside commercial users:

• $120.00 / hour assisted use
• $80.00 / hour unassisted use

Policies:

All users of the confocal microscope must be properly trained by the confocal microscope technician (“assisted” use) prior to being allowed to use the scope without assistance (“unassisted” use).

Procedures for turning on the scope and turning off the scope are located in a folder, which can be found in the confocal room.  Sign-up sheets can also be found in the same folder, and each user must fill in one of the sheets before leaving the room/microscope – the user must provide a legitimate billing account number on the sign-up sheet.

Every user must provide their own form of information storage in the form of a jump/thumb drive.  The hard disc space on the PC workstation may occasionally be “cleaned” and purged of user data; long-term storage of data cannot be guaranteed.  It is the responsibility of the user to take with them their own scan data when their session has ended.

No eating or drinking are allowed in the room at any time.

The core asks that proper acknowledgements be given in professional manuscripts and journal articles when data obtained from our facility is used for publication purposes.