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Biomedical Sciences

Quillen College of Medicine

Confocal Microscopy

Confocal Microscopy

The Confocal Microscope facility is located in the James H. Quillen College of Medicine, on the VA campus of East Tennessee State University in Johnson City, TN, in Building 119, Room 149D.  This location provides ease of access to the College of Medicine and the entire ETSU community, as well as to all interested parties outside of the ETSU family.

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 King-Rotator.

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

Highly efficient spectral separation is achieved via Leicas 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.

 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 HP2620 workstation computer:

Loaded with Windows 7-Professional (64 bit) operating system

Intel 8-Core Xeon 2.66 GHz processor

NVIDIA Quadro K600 1 GB high performance GPU

128 GB SATA SSD (solid state disc)

2 TB SATA hard disc drive

30 inch high brilliance LCD flat screen, true color, 2560 x 1600 pixel monitor

Leicas latest LAS X software package and 3D imaging software from Imaris, Inc.

The core also provides a state-of-the-art stand-alone Alienware computer 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:

Reservations for use of the confocal are done online via the university's email server Microsoft Outlook.  The technician will show you how to sign up online for using the confocal.  For all outside users, simply 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. 

Logging onto the Confocal Usage Sign-Up Calendar

These instructions describe how to open the calendar that is used for signing up to use the confocal microscope.  The instructions are for those who use PC computers only instructions for those who use Mac computers will be forthcoming on this web site. 

  1. Open Microsoft Outlook (your ETSU email provider).
  2. On the left hand side of the page, you should see a list of directories, subdirectories, and folders (such as Inbox, Sent Items, Deleted Items, etc.).
  3. If you do not see the Public Folders listed lastly, then proceed to the next step.  If you do see it listed, then proceed to step 6.
  4. At the bottom of that section, you will notice several navigation options on the navigation tool bar (Mail, Calendar, People, Tasks, Notes, Folders, Shortcuts, and one with three dots on it may be among those you see).
  5. If the Folders option is not visible, then click on the one with the three dots.  You will see other options appear (Notes, Folders, and Shortcuts).  Click on the option Folders.  This will put the Public Folders folder at the bottom of your list of directories.
  6. Click on the Public Folders bullet to open its subdirectories.  Then click on the All Public Folders bullet.
  7. Scroll down the list of subfolders and click on the Medicine bullet.
  8. Scroll down the list of subfolders and click on the Pharmacology bullet.
  9. You will see four items listed under Pharmacology, the first being Confocal sign-up.
  10. Click on Confocal sign-up and the sign-up calendar will open.
  11. Click on the time desired and list your name and lab name (PI), and lab phone number.
  12. The mouse cursor can be used to stretch the highlighted area downward to include the desired time for your session (from the center of the bottom of the highlight box).
  13. You can also scroll to other days or weeks on the calendar to sign up for future days sessions.
  14. When finished signing up, simply close out the folders and your information will be retained for other users to see when they log on to sign up.

The Outlook Calendar may look different when opened up on different computers, but can be configured in several ways (to show each day, each week, or by the month); this is done via the options listed on the tool bar at the top of the page.  The configuration is irrelevant as long as you list your information as mentioned above and close out the program.

Please call or email the confocal laboratory technician for any questions regarding this procedure.

Usage fees:

                For all ETSU users and other educational entities:

                                $60.00 / hour assisted use

                                $40.00 / hour unassisted use

                For all other outside commercial users:

                                $120.00 / hour assisted use

                                $80.00 / hour unassisted use

All users will initially be charged as assisted users until the confocal technician is assured that the user is skilled and confident enough with their use of the microscope to warrant unassisted use (this may require several repeated sessions as necessary).

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 either in the form of a jump/thumb drive or a DVD-R (or CD-R) disc.  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 acknowledgments be given in professional manuscripts and journal articles when data obtained from our facility is used for publication purposes.

 

 

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