Cellular Imaging Core

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

Technical Director: Mike Kirber
office: 617-638-7153
cell phone: 617-571-3408
e-mail: mkirber@bu.edu

Use of the devices/equipment in this core is by request and organized by an online scheduling program. To gain log-in credentials, please follow this link to complete the online application form.

Instruments/Services

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.

Rates

Spinning disk Perkin Elmer confocal
650 Albany Street, basement

Schedule this device (Log-in credentials required)

• This system is good for:
  • Time lapse of fast events such as translocation, electrophysiological events
  • Fixed or living samples
  • Subcellular imaging
    
    
• This system is not good for:
  • UV excited dyes
  • Very high resolution (less than 1 micron resolution)

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

Overview
The excitation source is a Krypton Argon Ion laser with lines at 488, 568, and 647 nm.  The system is suitable for FITC, Alexa 488, GFP, Rhodamine, Alexa 568, CY5, and Alexa 647.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. 
• Users wishing to use this piece of equipment without supervision will need to have annual laser safety training.
• Maintenance involves laser alignment, cleaning optics, and replacing the mercury arc 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.

Rates

Two-photon confocal
650 Albany Street, basement

Schedule this device (Log-in credentials required)

• 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.

Rates

Nikon deconvolution wide-field Epifluorescence system
650 Albany Street, basement

Schedule this device (Log-in credentials required)

• This system is good for:
  • Fixed samples or samples that survive at room temperature
  • Colocalization studies
  • Subcellular resolution (organelle imaging)
  • Generating a confocal image as well as 3D reconstruction
  • Low intensity fluorescence
    
    
• This system is not good for:
  • Recording events in living cells
  • 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 3 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

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.

Rates

Improvision monochromator wide-field deconvolution epifluorescence microscope
650 Albany Street, basement

Electronic scheduling not available at this time.

• This system is good for:
  • Fixed samples or samples that survive at room temperature
  • Colocalization studies
  • 1-2 micron resolution
  • Generating a confocal image as well as 3D reconstruction
  • Low intensity fluorescence
    
    
• This system is not good for:
  • Recording events in living cells
  • Very high resolution (less than 1 micron resolution)
  • Fast quenching dyes
  • Inexperienced users and infrequent users (this system requires longer training)

Function of Instrument
This system like the Nikon system above is a wide-field fluorescence system with the ability to take multiple images at different focal planes and use that information to remove out of focus fluorescence.

Overview
This system has the advantage of using a high-speed monochromator for the excitation, which can switch excitation wavelengths in several milliseconds.  The imaging device is a cooled CCD camera.  Several frames per second can be acquired. The system has a 3 wavelength dichroic and emission filter suited for DAPI (Also Hoechst and Alexa 350), FITC (also GFP, Alexa 488), and Rhodamine (also Alexa 568).  In addition a second filter set is available for Texas red (and Alexa 594). For most fluorescence work a 100x oil-immersion objective is used which is connected to a piezoelectric focusing drive.  A 40x long-working distance objective can also be used.

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.
• The software requires training and practice prior to use on experimental samples of value.
• Maintenance involves calibrating the monochromator, changing and aligning the xenon arc lamp, and cleaning the optics.
• New users should schedule a training session of 2 hours. Please book your training 2 weeks in advance. Bring a fixed sample to training and we will acquire images with you.

Rates

Zeiss LSM 710-Live Duo scan
650 Albany Street, basement

Schedule this device (Log-in credentials required)

Function of Instrument
This system makes use of patterned illumination and a unique linear CCD detector to obtain very high-speed (over100 frames per second at 512x512 resolution) confocal images.

Overview
This system can be combined for unique experimental challenges such as simultaneous uncaging and high-speed 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. More details will become clear when the instrument is set up.
• It will be covered by a service contract, but will require some routine cleaning and alignment.

Cyntellect--Laser Mediated Transfection , Purification and Manipulation of Cultured Living Cells for High Throughput Biology
650 Albany Street, basement

Schedule this device (Log-in credentials required)

Overview
Automated High-Throughput Laser-Mediated Cell Purification Coupled with Cell Imaging on the LEAPT System LEAP is an automated imaging and laser-based processing system for in situ analysis and manipulation of cells in culture. Based on robust semiconductor manufacturing technologies, LEAP provides unique benefits to biological researchers working with adherent or non-adherent cell types. LEAP enables high-throughput in situ imaging with multiple fluorescence and brightfield morphologic capabilities used for cell identification and classification. Targeted cells can then be automatically laser-irradiated to achieve a pure population of desired cells, (with high-yield and purity) and/or transfection (with high efficiency and low toxicity) in thousands of cells per second. LEAP has been used to enable extremely efficient purification of valuable cells in samples ranging from 101-107 cells. Applications include purification of adherent and non-adherent cells with labile surface markers, automated embryonic stem cell passageing, generation of uniform sized embryoid bodies, high-throughput cell line development, and cell cloning based on levels of protein secretion, and siRNA transfection into difficult cell types, will be presented.

Olympus stereo fluorescence imaging microscope
650 Albany Street, basement

Schedule this device (Log-in credentials required)

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. The software has a deblurring algorithm which corrects for much of the out of focus light which results in fluorescence image blurring in thick specimens. A feedback controlled temperature and CO2 microincubation system allows for time-lapse embryo or organ culture experiments.

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.