Elastin in an artery wall viewed with Multiphoton Excitation
Left: A single input photon (blue) excites a single photon (green). Right: Multiphoton Excitation. Two input photons (red) combine to excite a single photon (green).
Collagen in a rat tail tendon viewed with SHG
Second Harmonic Generation
CARS Levels

Multiphoton imaging and spectroscopy laboratory

Imaging mechanisms

Our facility combines three multiphoton contrast mechanisms in a single microscope, offering unrivalled flexibility allowing stain-free imaging of multiple species within a sample. As well as providing non-invasive image contrast, Multiphoton interactions also contain spectroscopic information which can provide novel mechanisms for detection and characterisation of biomolecules in-vivo.

Multiphoton Excitation

MPE is the most simple form of Multiphoton Microscopy (MPM) which is used to excite fluorescence. A pulsed laser is used to provide simultaneous excitation with two (or three) photons of low energy, exciting the fluorophore to the same level as one high energy photon. MPE has many advantages over normal laser scanning microscopy. The use of lower energy photons (infrared) reduces damage to the sample and also permits deeper penetration into scattering samples. Multiphoton excitation occurs only at the focal plane, removing the need for pinhole apertures.

As well as excitation of extraneous fluorophores, MPE can be used to excite indigenous tissue components such as NAD(P)H, flavins, and porphyrins.

Second Harmonic Generation

SHG is a second-order nonlinear optical process, which requires an environment without a centre of symmetry to produce signals. Two photons (ωp) are mixed in the sample to generate a third photon (2ωp) (see diagram below-right). Several indigenous tissue components are known to exhibit SHG which can be used to generate label free images.

Collagen fibres are a well-known source of SHG; the picture below-right shows an SHG image of collagen fibres within a rat tail tendon. SHG deposits no energy into the sample due to the emitted SHG photon energy being equal to the total absorbed excitation photon energy.

Coherent Anti-Stokes Raman Scattering

CARS is the most recent MPM imaging contrast. CARS microscopy derives contrast directly from Raman- active vibrational modes within molecules and requires two synchronised laser pulses of different wavelengths. CARS is a third order nonlinear optical process in which a pump field Ep and a Stokes field Es interact with a sample to generate a signal field Eas at the anti-Stokes frequency of ωas = 2ωp - ωs. When ωp - ωs is tuned to be resonant with a molecular vibration (&Omega), the CARS signal can be significantly enhanced, producing a vibrational contrast.

Unlike spontaneous Raman scattering, CARS produces a highly directional field. The two excitation beams (ωp and ωs) form a beating field with frequency ωp - ωs. When ωp - ωs matches &Omega all the molecules within the interaction volume vibrate in-phase.

Advantages of CARS

CARS microscopy permits biological imaging with several advantages:

  • Raman resonance enhancement provides chemical selectivity without the need for labelling. 
  • There is little scattering of the near-infrared excitation beams, allowing deep penetration in tissues.
  • Due to the anti-Stokes shift, the CARS signal is of shorter wavelength than one-photon fluorescence. This allows detection in the presence of a strong fluorescent background.
  • Coherent addition of CARS fields generates a large signal.
  • Nonlinear dependence on excitation intensities produces inherent 3D resolution.
  • Low absorption of the near-infrared excitation beams, significantly reduces the photodamage in biological samples.
Coherent Anti-Stokes Raman Scattering (CARS)