Advertisement

Every Photon is Sacred

If you liked confocal microscopy, you're going to love multi-photon systems, now available from Bio-Rad Instruments. Taking the principle of confocal microscopy one giant step further, multi-photon microscopes use rapidly oscillating, low energy laser light to illuminate a small sample area--as small as 0.1 femtoliter. Working cooperatively, the energy from two (or three in some cases) photons can excite fluorochromes that ordinarily require high energy excitation. What does this get you? Deep

June 22, 1998

If you liked confocal microscopy, you're going to love multi-photon systems, now available from Bio-Rad Instruments. Taking the principle of confocal microscopy one giant step further, multi-photon microscopes use rapidly oscillating, low energy laser light to illuminate a small sample area--as small as 0.1 femtoliter. Working cooperatively, the energy from two (or three in some cases) photons can excite fluorochromes that ordinarily require high energy excitation. What does this get you? Deeper sample penetration, photobleaching that is restricted to the small area of excitation, and less phototoxicity, allowing for real-time and continuous sampling of live specimens.


Comparison of confocal and multi-photon imaging depth penetration. XZ profiles through an acid fuchsin stained, monkey kidney pathology sample imaged through a depth of 140 µm with confocal (left) and multi-photon microscopy. Scale bar = 20 µm. (Courtesy of Victoria Centonze Frohlich, University of Wisconsin, Madison).
Confocal microscopy had become the standard for fluorescence microscopy since it allows for greater depth of imaging and more efficient signal collection than conventional fluorescence microscopy. While confocal microscopy works by restricting the signal collection to a small area, two-photon microscopy restricts the actual excitation to a small region. Unlike confocal imaging, in which the signal generated is directly related to the intensity of the laser light, with two photon imaging, which is a nonlinear process, the signal depends on the square of the incident light intensity. This limits the excitation to a point of focus rather than a plane, improving resolution and decreasing photobleaching.

According to Andrew Dixon, Scientific Director of Bio-Rad's microscopy division, "The defining practical difference between a conventional and two-photon system is the specification of the laser light source. Whereas a conventional system uses a light source emitting a continuous intensity of a few milliwatts, a two-photon system uses a laser emitting ultrashort pulses of light with peak intensities of kilowatts. It is this combination of very high power over a very short period of time that allows the 'two-photon' mode of fluorescence excitation to occur."

A further advantage of two-photon microscopy is that it produces optical section images deeper into the specimen than what can be obtained with confocal microscopy. The long red and infrared wavelengths used in two-photon imaging penetrate deeper into the sample than UV, blue, or green wavelengths typically used in confocal imaging.

In a licensing arrangement with the group at Cornell University that described and patented multi-photon microscopy systems (W. Denk et al., Science, 248:73-6, 1990), Bio-Rad has introduced the MRC-1024MP which is based on its MRC-1024 confocal system. The MRC-1024MP is available as a dedicated multi-photon system with a femtosecond-pulsed tunable wavelength TiS or fixed wavelength Nd:YLF laser or as a combined confocal/multi-photon system with an Ar or Kr/Ar laser. The MRC-1024MP system has the following components:

  • a modified scanhead with mirror coatings and optics that provide transmission of wavelengths between 690 nm and 1000 nm.
  • an optics box that allows for selection of neutral density filters to control laser power and allow com- bination of laser wavelengths.
  • provision for a number of subpicosecond laser options, combined with a conventional confocal laser source.
  • optional nondescanned detector option, which increases the sensitivity several-fold over that of conventional detectors.
  • LaserSharp software that has an image acquisition, analysis, and processing package with a physiology module.

Kathy Spenser, a Scientific Associate, and Martin Friedlander, Associate Professor at The Scripps Research Institute, La Jolla, Calif., are using the Bio-Rad two-photon technology in the development of gene delivery systems for correcting inherited and degenerative eye disorders, such as retinitis pigmentosa, macular degeneration and Stargardt's disease. The two-photon technology will allow Spenser to "optically section intact, unfixed retinas" to determine the expression of transfected, fluorescently tagged proteins. In conjunction with the precise stage controls of the conventional Bio-Rad confocal system, the longer wavelength of the two-photon laser allows her to literally step through the entire, thick specimen, saving her the trouble of having to manually section the eye and reconstruct a three-dimensional image.

In addition to the conventional uses of fluorescence imaging, scientists at the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana-Champaign have described two novel, what they term "interactive," uses of two-photon imaging. They are using two-photon microscopy to track single particles in order to study protein diffusion and interaction (the example they give is the diffusion of a fluorescent particle during macrophage endocytosis). In addition, they are using two-photon systems to initiate localized chemical reactions, such as photo-polymerization.

For further information, contact Bio-Rad at 800-4-BIORAD or visit the company web site at www.bio-rad.com.

Follow The Scientist

icon-facebook icon-linkedin icon-twitter icon-vimeo icon-youtube
Advertisement

Stay Connected with The Scientist

  • icon-facebook The Scientist Magazine
  • icon-facebook The Scientist Careers
  • icon-facebook Neuroscience Research Techniques
  • icon-facebook Genetic Research Techniques
  • icon-facebook Cell Culture Techniques
  • icon-facebook Microbiology and Immunology
  • icon-facebook Cancer Research and Technology
  • icon-facebook Stem Cell and Regenerative Science
Advertisement
Eppendorf
Eppendorf
Advertisement
The Scientist
The Scientist