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The Light Microscopy facility is accessible 24 hours a day, seven days a week to Janelia scientists. To maintain a safe and supportive shared resources environment, please refer to the following (minimal) guidelines. We also provide help with writing M&M sections and Acknowledgements. If you have questions about the facility and/or how to schedule time on the instruments, contact Damien Alcor or Michael DeSantis.

Access/Scheduling

Training by a staff member is required to gain access to the microscopes. Once completed, you will have permission to reserve the appropriate instruments using the Resource Scheduler.

Fees are applicable: Please see the fee structure, below.

  1. Training:
    • If you do not have permission to use a microscope, please contact us.
    • We strongly encourage users to discuss their experiment and goals first to ensure that the intended instrument is suitable to their needs.
    • In your email, use the Resource Scheduler to suggest times for a two-hour training session on the intended instrument (based on microscope availability).
    • Please bring with you an expendable sample to be imaged - ideally, it should be similar to what you eventually want to study; if necessary, the facility can provide samples for training.
    • A second, supervised training session may be required.
  2. Safety and responsibilities:
    • Please exercise appropriate lab safety, following all Janelia rules and safety regulations especially when working with chemicals and live samples. Food or drinks are prohibited near the microscopes.
    • Be careful switching and handling of objectives. When focusing a sample, users should be aware of the proximity of the objective lens to the stage.
    • Leave the microscopes and rooms clean after use. Clean any oil/water-immersion objectives used.
    • If you are the last scheduled user of the day, please shut down the equipment in accordance with the operating procedures, otherwise try to keep lasers in standby mode to prolong their life.
    • Please report any problems to staff personnel as soon as possible.
  3. Sign-up and usage rules:
    • For trained users, always reserve instruments using the Resource Scheduler.
    • Unscheduled use is strongly discouraged. We maintain an open and honest working environment trusting users to schedule instrument time to reasonably reflect actual use.
    • Please be considerate to the community and do not monopolize equipment for days at a time (contact a staff member if you have been unable to reserve time as a result).
    • Reservations may be canceled without penalty up to two hours prior to the start of the scheduled booking. Afterwards, reservations can no longer be modified; to extend the current session, simply create a new reservation (making sure to select the appropriate resource). After the scheduled booking, any significant changes can only be made by contacting a staff member in a timely manner.

Data Storage

Please refer to the following guidelines regarding data storage at the Light Microscopy Facility. Read it carefully. The facility is not responsible for lost data.

  1. Storage on acquisition computers and workstations:
    • Data should only be saved in the “USER_DATA” directory and within your lab/username folder.
    • To maintain free storage space, data should eventually be moved from the local drive. Cleanup will be done on a regular basis such that data older than one month may be deleted.
    • Data stored outside of the USER_DATA folder may be deleted without notice.
    • It is the user’s responsibility to transfer their data accordingly within this time window. Users are strongly encouraged to move their data directly to their lab’s dm11 share space at the end of each imaging session.
  2. Preserving “Settings” files on acquisition computers:
    • We understand that it is useful to keep some small files on these computers in order to reuse acquisition settings/parameters. Please save them in the "USER_Settings" directory. This folder won't be cleaned up automatically but we will ensure that it is not used for data storage.
  3. Storage on \\dm11\imaging:
    • This shared space should be used only temporarily. It has limited storage and data older than one month will be deleted when used storage reaches saturation. We will send out a notification email one week prior to cleanup. Users should ultimately transfer data to their lab’s dm11 share space.
    • Connecting to the Imaging Server:
                      Map Network Drive
                      Folder: \\dm11.hhmi.org\imaging
                            user: hhmi\imaging
                            pswd: .Janelia (don't forget "." and capital "J")

Fee Structure

Usage of the following instruments is automatically billed via the Equipment Scheduler Reservation system:

  1. Zeiss LSM 880 with Airyscan, LSM 800, and LSM 880 NLO confocal microscopes: $30/hr
  2. Slide scanners (Pannoramic and TissueGnostics): $30/hr
  3. Nikon Eclipse Ti widefield microscope: $30/hr
  4. Zeiss Lightsheet Z.1 microscope: $30/hr
  5. Lattice Light-sheet microscopes: $35/hr
  6. Zeiss Axio Imager.Z1 with ApoTome.2 widefield microscope: $15/hr
  7. Olympus MVX-10 macroscopes: $0/hr
  8. Olympus BX51 compound microscope: $0/hr
  9. Workstation #1 (Imaris) and Workstation #3 (Strataquest): $10/hr (all other workstations: $0/hr)

A half-fee discount is applicable to the following instruments during night usage (6pm to 8am):

  • Zeiss LSM 880 NLO confocal microscope
  • Zeiss Lightsheet Z.1 and Lattice Light-sheet microscopes when reserved for several days continuously (contact us)

Additional fees are assessed for staff support:

  • Training: $60/hr
  • Technical assistance, image processing/analysis: $60/hr

How to write Materials & Methods sections and Acknowledgements

In order for other researchers to understand and replicate your findings, it is necessary to thoroughly document both your experimental setup and how the data was acquired. There are many aspects to relate including basic microscope equipment information (see equipment) in addition to some of the criteria listed below (adapted from NIC at HMS). Please ask us if you are unsure or have any questions.

  1. Make and model of microscope (e.g., Zeiss LSM 880)
  2. Description of advanced imaging modality – confocal, TIRF, super-resolution, etc. (e.g., Zeiss Airyscan)
  3. Hardware or software auto-focusing, if used (e.g., Nikon perfect focus system)
  4. Type, magnification, and numerical aperture of objective lenses (e.g., Zeiss Plan-Apochromat 63x/1.4 Oil)
  5. Imaging environmental conditions – chamber, cell culture (for live samples) or mounting media, temperature, buffers, etc.
  6. List of fluorophores.
  7. Fluorescent filters, including peak transmission and bandwidth (e.g., Chroma 490/30 excitation filter and 525/50 emission filter)
  8. Laser type, line, and selection method used (e.g., 488nm line from an argon laser, selected with a 488/10nm filter versus 488nm line from an argon laser, selected with an AOTF)
  9. Make and model of camera (e.g., Hamamatsu ORCA-Flash4.0 V3 sCMOS camera)
  10. Other motorized components (e.g., Märzhäuser Wetzlar 130x100 STEP linear encoded stage for tile scans)
  11. Acquisition software make and version (e.g., ZEN Black 2.3 SP1)
  12. Image analysis software and programming tools detailing any image processing algorithms employed

It is best to document each microscope component (e.g., camera, objective lens) used during your experiment. You are encouraged to send a draft of your Materials & Methods section to the Light Microscopy staff – we will be happy to edit to ensure accuracy. Lastly, if you collected images at the facility, all users should cite the Light Microscopy Facility accordingly in the Acknowledgements section of your manuscript. When staff has helped beyond basic training and provided valuable input, it may also be appropriate to include authorship.

Here are examples of a complete Materials & Methods section and Acknowledgements:

Cells were grown on No. 1.5 coverslips and mounted in a [INCUBATOR (stage top or enclosure)] heated chamber warmed to X°C. [TISSUE CULTURE MEDIA and BUFFER] without phenol red was used during image acquisition. All images were collected using [IMAGING MODALITY and INVERTED/UPRIGHT MICROSCOPE] equipped with [OBJECTIVE LENS Correction Mag/NA] using the definite focus system to maintain focus over time. [FLUOROPHORE] was excited with a [LASER LINE] nm laser line from a [LASER SOURCE/TYPE] selected using an [EXCITATION FILTER or AOTF]. The measured laser power and irradiance at the sample were [POWER] mW and [IRRADIANCE] kW/cm2, respectively, and fluorescence was detected using a [DICHROIC MIRROR] beamsplitter and a [EMISSION FILTER] emission filter. Images were acquired with a [CAMERA] controlled with [ACQUISITION SOFTWARE] software. For time-lapse experiments, images were collected every X min using an exposure time of X ms without camera binning. At each time point, acquisitions were made at multiple stage positions via a [MOTORIZED STAGE] such that a z-stack comprising X slices (step size of X microns) was performed using the [FOCUS MOTOR]. Z-series are displayed as maximum intensity projections; brightness and contrast were adjusted (identically for compared image sets) using [IMAGE ANALYSIS SOFTWARE].
We gratefully acknowledge the shared resources and project teams at Howard Hughes Medical Institute (HHMI) Janelia Research Campus (JRC). [IMAGING MODALITY] imaging experiments were conducted at the JRC Light Microscopy Facility. We would like to thank [DAMIEN ALCOR/MICHAEL DESANTIS] for [MICROSCOPY TRAINING] and/or their assistance with [IMAGING/IMAGE ANALYSIS].

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