Two-Photon Microscopy, Non-Linear Optics (NLO)

A two-photon microscope is similar to a confocal microscope. Both provide optical sectioning and are very well suited for 3D imaging. In both systems a laser beam is focused to a small spot and scanned line-by-line over the sample.

In a confocal microscope, all dye molecules along the beam-path are excited (and eventually bleached). Out of focus light is rejected using a pinhole.

In a two-photon microscope, only dye molecules within the focal spot are excited, thus there is no need for a pinhole. This fact and the use of near-infrared-light (NIR) allows imaging of thicker biological samples.

Two-photon microscopes use near-infrared (NIR) light to excite dyes normally excited with shorter wavelengths in one-photon microscopy. Most tissue is transparent to NIR light. NIR light is reported to be less harmful to live cells and developing animals.

The two-photon effect can be described in terms of non-linear optics (NLO). The terms NLO, two-photon microscope or multi-photon microscope (MPM) are often used interchangeably, even if their meanings are not identical.

At the Stowers Institute we have three two-photon microscopes.

Stowers Links:
Image Center: Instruments
Adv. Instr. & Physics: Confocal Laser Scanning Microscope (LSM)
Foundations of Microscopy Seminar: Non-Linear Optics

Imaging in 3D: Confocal vs. NLO Microscopy: View Presentation/Download Presentation (zip)

Web Links:
Molecular Expressions: Multiphoton Fluorescence Microscopy
User group: Multi-Photon Laser Scanning Microscopy
W.W.Webb Group at Cornell University

Literature:
Molecular Expressions: Multiphoton Fluorescence Microscopy - Selected Literature
Denk, W., J. H. Strickler, et al. (1990). "Two-Photon Laser Scanning Fluorescence Microscopy." Science 248(4951): 73-76.Download PDF
M. Dickinson: Multiphoton Laser Scanning Microscopy: Introduction into Theory and Operation
Koenig, K. (2000). "Multiphoton microscopy in life sciences." Journal of Microscopy, 200(2): 83-104Download PDF
Mertz, J. (2004). "Nonlinear microscopy: new techniques and applications." Curr Opin Neurobiol 14: 610-6.Download PDF

In one as well as two-(or multi-) photon fluorescence, a dye molecule emits light when an electron relaxes from an excited energy state to it's ground level. The energy difference between the two levels (electronic and vibrational states) determine the emission spectrum. It is identical for one- and multi-photon excitation.

The electron moves from the ground level to the excited level by absorbing one or multiple photons. Typically this state is a vibrational state and the electron dissipates energy before emitting a photon and relaxing to the ground level. The sum of the energies of all absorbed photons has to be larger than the energy gap and is larger (or equal) to the energy of the emitted photon.

In one photon fluorescence, this translates into the absorbance of one photon with a shorter wavelength than the emitted photon.

In two-photon fluorescence two photons with about double the wavelength (half the energy) are absorbed.

Stowers Links:
Adv. Instr. & Physics: Fluorescence

For two-photon excitation, two photons have to be absorbed by the dye molecule at the same time (within about 15 fs). This requires an extreme high photon flux, only present in the focus of a high numerical aperture lens using a strong, pulsed near infrared laser. This is shown in the image. In the upper part of the curvet, all molecules within the beam-path are excited, while in the lower part only molecules within the focal spot emit light.

The optical sectioning capability of a two-photon microscope is based on this fact - only dye molecules within the in-focus plane are excited. Thus, contrary to a confocal microscope, a two-photon microscope does not need a pinhole.

Two-photon excitation follows different selection rules than one-photon excitation. Thus one can not just shift the one-photon excitation spectrum to twice the wavelength. Typically two-photon spectra are shifted to shorter wavelengths than expected by doubling the wavelength of one-photon spectra. Two-photon spectra are in most cases much broader than one-photon spectra.

Good results have been reported for the following excitation wavelengths:

Unfortunately not many two-photon excitation spectra are published. Reported excitation wavelengths are often unreliable. We typically use excitation fingerprinting to determine the best imaging conditions.

Web Links:
Carl Zeiss CellScience: Multi-photon Spectra
Cornell University (Ward W. Webb): 2P cross sections

Literature:
Fisher, J. A. N., B. M. Salzberg, et al. (2005). "Near infrared two-photon excitation cross-sections of voltage-sensitive dyes." Journal of Neuroscience Methods 148(1): 94-102.Download PDF
Xu, C., W. Zipfel, et al. (1996). "Multiphoton Fluorescence Excitation: New Spectral Windows for Biological Nonlinear Microscopy." PNAS 93(20): 10763-10768.Download PDF

Most animal cells and tissues are considered to be nearly transparent in the spectral range from about 700 nm to 1100 nm owing to the lack of efficient one-photon absorbers. This enables deeper penetration of the NIR excitation laser-beam into tissue. Damage to the sample is reduced.

The graphic shows absorption spectra of major intracellular absorbers. The molecular exctinction coefficients of oxygenated haemoglobin and melanin and the absorption coefficient of water are shown.

Literature:
Koenig, K. (2000). "Multiphoton microscopy in life sciences." Journal of Microscopy, 200(2): 83-104Download PDF

Most components of a confocal laser scanning microscope and a two-photon system are identical. All two-photon microscopes at the Stowers Institute can be used in addition as confocal microscopes with excitation wavelengths in the visible range.

The pulsed NIR laser light is coupled into the confocal detection module. It is directed toward the sample using a short-pass filter (e.g. KP 700). Two mirrors scan the laser-beam over the sample. The detection head is attached to a standard microscope. The beam is focused into the sample with a high numerical aperture lens optimized for near-infrared transmission and minimum dispersion.

Fluorescence is collected using the identical lens. The fluorescence light hits again the scanning mirrors. Depending on the position of the scanned excitation laser beam, the fluorescence is generated at different locations inside the sample. After the fluorescence light passes the scanning mirrors, it is stable. This beam path is called the de-scanned detection path. Because the fluorescence in a two-photon setup has a shorter wavelength than the excitation laser-beam, it can pass the short-pass filter. The light passes through the pinhole. In most two-photon applications the pinhole is opened to it's maximum size. The light is detected using one or multiple photo-detectors.

The de-scanned detection path is not the most efficient one to detect light scattered out of focus. It is more efficient not to route the fluorescence light back into the detection head and not to let it pass the scanning mirrors. A short-pass filter close to the sample (typically in the filter turret of the light microscope) reflects the fluorescence light directly onto a detector. Because now the light is moving in synchrony with the excitation laser beam, the detector has to be large enough and no pinhole can be used.

In some cases forward fluorescence or transmitted light is collected using the condenser of the microscope.

Stower's Links:
Adv. Instr. & Physics: Confocal Laser Scanning Microscope (LSM)
Image Center: Instruments
Ron Yu Lab

Web Links:
Carl Zeiss Laser Scanning Microscopes

Literature:
Manual LSM 510 META 3.5

To achieve the photon density necessary for two-photon excitation pulsed near-infrared lasers are used. Typically they are diode pumped Ti:Sapphire lasers. At the Stowers Institute we are using Coherent Chameleon lasers. The wavelength can be tuned and the laser is controlled by the microscope software.

Web Links:
Coherent Chameleon Ultrafast Ti:Sapphire Lasers
Molecular Expressions: Ti:Sapphire Mode-Locked Lasers

Literature:
Coherent Chameleon Ultra : Manual. Chapter 7 is a good introduction into the physics of pulsed lasers.
Chameleon Data sheet Download PDF
Chameleon Ultra Data sheet Download PDF