Cellular Imaging Core

Scientific Director: Orian Shirihai
e-mail: shirihai@bu.edu

Technical Director: Mike Kirber
office: 617-638-7153

e-mail: mkirber@bu.edu

Introduction

We are an optical microscopy core facility with a range of digital imaging microscopy systems for studying cells and tissues. Most of our equipment is located in the basement (B15) of the EBRC building at 650 Albany St.

Scheduling and pricing information are available at http://coremgmt.bumc.bu.edu


IonOptix calcium imaging system

650 Albany Street, basement

  • This system is good for:
    • Living cells
    • Calcium imaging
    • Time lapse
  • This system is not good for:
    • Subcellular resolution
    • Inexperienced users and infrequent user (due to relatively long training time)

Function of Instrument
This system is designed primarily for time-lapse high-speed imaging of intracellular calcium in living cells using Fura 2.

Overview
The system uses an intensified CCD camera and chopper wheel to rapidly switch between 340 and 380 nm excitation. The software allows for background correction and easy calibration so that numerical values as well as images of calcium concentration in time can be obtained. The system can yield 30 ratiometric calcium images per second.

Training and Usage and Maintenance

  • Training involves familiarizing the user with the appropriate turn on and turn off sequences so as not to damage certain instrumentation components.
  • Instruction will include use of the IonWizzard software, as well as calcium calibration and methods of background fluorescence subtraction.
  • The major maintenance issues involve replacing the arc lamp and mechanical shutters, which tend to fail.
  • New users should schedule a training session of 2 hours. Please book your training 2 weeks in advance.

Olympus DSU spinning disk confocal microscope

650 Albany Street, basement

  • This system is good for:
    • Time lapse of fast events such as protein translocation.
    • Fixed or living samples
    • Subcellular imaging
    • DAPI, FITC, Alexa 488, TEXAS red, Alexa 594, Alexa 647, Cy5 and similar indicators.
    • Calcium imaging using fura 2 (non-confocal)
  • This system is not good for:
    • Very high resolution (less than 0.5 micron resolution)

Function of Instrument
This system is a relatively high-speed (up to 11 frames / sec), medium sensitivity confocal.

Overview
The excitation source is a Prior Lumen pro 220 mercury halide wideband light source. The detector is a cooled CCD camera. It is ideal for capturing high-speed events such as calcium transients, rapid translocation events, and transient membrane potential changes. It is also suitable for fixed samples.

Training and Usage and Maintenance

  • Training for the use of this system involves familiarizing the user with the proper turn on and turn off sequences so as not to damage the equipment.
  • The software is somewhat complex and requires training.
  • Maintenance involves optical alignment, cleaning optics, and replacing the light source bulb.
  • New users should schedule a training session of 2 hours. Please book your training 2 weeks in advance. Bring your sample to training and we will acquire images with you.

Two-photon confocal

650 Albany Street, 7th floor (712)

  • This system is good for:
    • Living cells
    • Time lapse of fast events such as translocation, electrophysiological events
    • Fixed or living samples
    • Imaging at subcellular resolution
    • UV excitable dyes
  • This system is not good for:
    • Red excitable dyes
    • Use of two dyes with similar emission wavelength (this system is better for single excitation dual/triple emission)

Function of Instrument
This system uses pulsed infrared light to excite dyes normally excitable in the visible and UV range. It uses a single point scanning system and multiple photomultiplier tubes to detect 3 ranges of emitted light simultaneously.

Overview
The advantages of this system are that the infrared light is well tolerated by living cells, the infrared penetrates living tissue better than visible or UV (~1mm depth), out of plane bleaching is minimized, and 3 emission wavelengths can be collected simultaneously. It is well suited for live cell and tissue imaging. The disadvantages of the system are that it is slow (several seconds per frame), it can be difficult to excite a single dye independently of others in the preparation, and it is not well suited for very low fluorescence experiments.

Training and Usage and Maintenance

  • This system was developed in house and has exposed class IV laser beams. It therefore needs to be used with an operator.
  • There are also sensitive photomultiplier tubes which can be destroyed with improper use.
  • Regular users should have annual laser safety training.

Maintenance involves laser alignment, optical path alignment, cleaning of optics, and replacing mirrors in the light path and scanning optics. Images obtained from this instrument can be read with NIH ImageJ.

Nikon deconvolution wide-field Epifluorescence system

650 Albany Street, basement

This system is good for:

  • Fixed samples or live cell studies
  • Colocalization studies
  • Subcellular resolution (organelle imaging)
  • Generating a confocal image as well as 3D reconstruction
  • Low intensity fluorescence

This system is not good for:

  • Very high Z axis resolution
  • Fast bleaching dyes

Function of Instrument
This system uses a broadband mercury-halide light source and sets of high-throughput filters to excite fluorescent dyes and proteins.

Overview
At present we have 4 filter sets including:

  1. UV excitation and blue emission for DAPI, Hoechst, and Alexa 350
  2. Blue excitation green emission for FITC, GFP, and Alexa 488
  3. Yellow-green excitation red emission for Texas RED, Alexa 594, and mcherry
  4. red excitation far red emission Alexa 647 (cy5)

The system has motorized filter changer, nosepiece and focusing drive which allows for automated image acquisition. All imaging parameters are stored in the saved files for later analysis. The focus drive allows for stacks of images at different focal planes to be taken and that information is used to remove out of focus fluorescence and confocal like images to be acquired (deconvolution). The imaging device is a cooled (-30 degrees C) sensitive CCD camera. This system is well suited for low intensity fluorescence samples.

Training and Usage and Maintenance

  • Training involves proper turn on and turn off sequences.
  • It also involves learning the proper operation and cleaning so as not to damage the optics or other parts of the instrument.
  • Instruction in the use of NIS Elements software is fairly straightforward.
  • Generally, the instructor is operating the instrument for the first session and explaining as the experiment progresses and is present for the next experiment if there are questions.
  • Maintenance involves cleaning the optics, adding and removing filter cubes for particular experiments and changing the mercury halide lamp.
  • New users should schedule a training session of 2 hours. Please book your training 2 weeks in advance. Bring your sample to training and we will acquire images with you.

Zeiss LSM 710-Live Duo scan

650 Albany Street, basement

Function of Instrument

This system is an advanced confocal microscope with a variety of capabilities well suited for live-cell as well as fixed specimen imaging.

Overview
This system makes use of patterned illumination and a unique linear CCD detector to obtain very high-speed (over100 frames per second at 512×512 resolution) confocal images. It also has an advanced point-scan confocal capability for visible and 2-photon excitation.  The point scan system has allows the user to seleect 3 customizable portions of the emission spectrum and steer each to a separate photomultiplier tube.  This instrument can be used for unique experimental challenges such as simultaneous uncaging and high-speed live-cell imaging.

Training and Usage and Maintenance

  • This instrument will require significant training to understand and use properly.
  • It is actually the combination of 2 instruments one which is high-speed and uses patterned illumination and the other which is slower and uses scanned point illumination.
  • People who are not regular users will require an operator assistance.
  • The slower point scan system also has 2-photon excitation capability.
  • The use of this instrument will require annual laser safety training.
  • It will be covered by a service contract, but will require some routine cleaning and alignment.

Olympus stereo fluorescence imaging microscope

650 Albany Street, basement

Function of the instrument

This system is a low magnification white light and fluorescence upright imaging microscope suitable for imaging embryos, organs and tissue samples.

Overview
This system is a low magnification stereo microscope ideal for imaging live embryos and whole organs. It has a computer controlled motorized z-axis drive for the automated acquisition of images in multiple planes. It uses a mercury-halide fluorescence light source and filters for fluorescence imaging of nuclear stains as well as expressed fluorescent proteins. The system is equipped with a cooled monochrome CCD camera for high-sensitivity fluorescence imaging. The camera can also be used for transmitted light and oblique illumination images. Color images can be obtained using an RGB LCD electronic emission filter.  A microincubation system allows for time-lapse embryo or organ culture experiments.

  • Training involves proper turn on and turn off sequences.
  • It also involves learning the proper operation and cleaning so as not to damage the optics or other parts of the instrument.
  • Generally, the instructor is operating the instrument for the first session and explaining as the experiment progresses and is present for the next experiment if there are questions.
  • Maintenance involves cleaning the optics and changing the mercury halide lamp.
  • New users should schedule a training session of 1 hour. Please book your training 2 weeks in advance. Bring your sample to training and we will acquire images with you.

 

Cyntellect Celigo microwell plate imager

650 Albany Street Basement

Function of the instrument

This instrument can do automated imaging and cell  classification and counting in standard multiwell plates.

Overview

  • Accepts T-flasks (T-25 and T-75) and most multi-well plates (1536-well to 6-well)
  • Extremely accurate brightfield cell imaging and identification across the entire well
  • Three channel fluorescence (in addition to brightfield)
  • Extremely rapid scanning (~5-15 min across a range of microplates)
  • Intuitive and easy-to-use, yet powerful, software segmentation and gating interface

Rules for Booking

  • Each lab can book a maximum of 10 hours of day time use or 15 hours of total use per week on each instrument.
  • This rule does not apply for “Next Day” reservation (booking of time for the coming day).
  • Therefore, frequent users are welcome to fill in the open gaps but they can book these hours only 1 day before the use.

Letters of Support: We are glad to provide letters of support for grants. Please email mkirber@bu.edu. If you give us ample time we would be glad to review sections related to your imaging experiments.

Help us help you.

Grants: The Cellular Imaging Core operates at a loss and is subsidized by the Department of Medicine. What does not get included in the balance sheets are the indirect costs generated from grants obtained with the help of data from our core. You can help us continue to serve you by letting us know when you submit a proposal and/or are awarded a grant which contains data obtained from the use of our Core.

Acknowledgments: We would greatly appreciate it if authors would acknowledge the Cellular Imaging Core in their publications containing data obtained with the equipment and/or assistance of Core personnel.