Laser Scanning Confocal Microscope

The LSCM is primarily used for imaging fluorescent samples. Organelles and proteins in cells or tissues are imaged using native fluorescence or by adding fluorescent tags. The LSCM is based on point scanning approach to image formation whereby the information for each pixel is obtained sequentially instead of the broad area excitation approach used for widefield imaging. A laser is used for selective excitation of a fluorophore and an optical bandpass filter limits the light reaching the detector. The LSCM is useful for three-dimensional reconstruction of data. Spatial resolution ~ 300 nanometers is achievable with objectives of numerical aperture of 1.3. The Zeiss LSM 410 was upgraded by LSM Technologies (optics, detectors, and computer). The instrument is on a floating optical table to minimize vibration transfer.

The LSCM is primarily used for imaging fluorescent samples. Organelles and proteins in cells or tissues are imaged using native fluorescence or by adding fluorescent tags. The LSCM is based on point scanning approach to image formation whereby the information for each pixel is obtained sequentially instead of the broad area excitation approach used for wide-field imaging. A laser is used for selective excitation of a fluorophore and an optical bandpass filter limits the light reaching the detector. The LSCM is useful for three-dimensional reconstruction of data. Spatial resolution ~ 300 nm is achievable with objectives of numerical aperture of 1.3.

Specifications

Excitation wavelength multi-line argon ion laser (457, 488, 514 nm)
three HeNe lasers (543, 594, 633 nm)
Filters Bandpass
Longpass
Objective lens 10x/0.3NA
20x/0.75NA
40x/1.3NA
63x/0.9NA (dipping objective)
100x/1.3NA
Simultaneous excitation 2 channels
488 nm and 594 nm (recommended)
Acquisition Time sequence
Differential interference contrast Using 10x and 100x objective lenses
Advances capabilities Objective inverter (LSM Technologies) for upright microscopy applications Widefield imaging, CCD camera as detector (broadband excitation and emission) ~405nm excitation (achieved using the ultra-fast laser system and SHG) FCS2 Temperature control chamber (contact Dr. Jim Xiang for use of the FCS2) Two-Photon Excitation Microscopy Fluorescence Lifetime Imaging Microscopy

Settings

User may excite fluorescence of elements within the sample, or add fluorescent tags. Excitation energy is selected by use of one, or two lasers simultaneously. Sequential colour-imaging, and z-sectioning procedures may be used to acquire images. Widefield images may be collected for comparison.
This system uses the Verdi / Mira / Pulse Picker / Harmonic Generator suite.

More Information

General statement for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. The images were acquired using a LSM410 (Zeiss, Thornwood, NY) upgraded by LSM Technologies (Etters, PA). An excitation wavelength of ___ nm from a multiline argon ion laser / HeNe laser was used and a bandpass/longpass emission filter (part # and manufacturer) were used for imaging. The total image acquisition time was ___ seconds and the pinhole was set to _____ airy units. The step size for three-dimension reconstruction was ____ micrometers and a total of ___ images were acquired.

Recommended Reading

  • Conn, P. Michael, ed. Confocal Microscopy. San Diego: Academic Press, 1999.
  • Diaspro, Alberto, ed. Confocal and Two-Photon Microscopy, Foundations, Applications, and Advances. New York: Wiley-Liss, 2002.
  • Paddock, Stephen ed. Confocal Microscopy (Methods in Molecular Biology Volume 122). Totowa, NJ: Humana Press, 1998.
  • Pawley, James B., ed. Handbook of Biological Confocal Microscopy, 2nd edition. New York: Plenum Press, 1995.
  • Periasamy, Ammasi and Richard N. Day, eds. Molecular Imaging: FRET Microscopy and Spectroscopy. New York: Oxford UP, 2005.
  • Yuste, Rafael, et al., eds. Imaging Neurons: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Lab Press, 1999.