Second Harmonic Generation (SHG)

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). 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. 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)

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.

Equipment

The front-end of our system is a modified Olympus IX71 inverted microscope. Dual sources, a pico-second OPO tunable from 700 – 1400nm (Levante Emerald from APE, Berlin), and a switchable femto- and pico-second system tunable from 700 – 1600nm (MIRA-D, and MIRA OPO from Coherent, USA) provide the multiple contrast modalities; Multiphoton fluorescence, Second- and Third-Harmonic Generation (SHG & THG) and Coherent Anti-stokes Raman Scattering (CARS).

Olympus FV300 and IX71 inverted microsope

• A modified Olympus FV300 and IX71 allows multiphoton imaging.

MIRA 900D

• Dual femto- and pico- second operation
• In femto mode provides excitation for second harmonic generation, multiphoton fluorescence, multiplex CARS and time-resolved CARS
• In pico mode provides excitation for frequency modulated CARS
• Output power: 1.2 W
• Wavelength Range: 690 - 990 nm
• Pulse width (femto mode): ~100 fs
• Pulse width (pico mode): ~1 ps

MIRA OPO

• Dual femto- and pico- second operation
• In femto mode provides excitation for third harmonic generation, sum frequency generation, pump-probe microscopy
• In pico mode provides excitation for frequency modulated CARS
• Output power: 300 mW
• Wavelength Range: 1050 - 1600 nm
• Pulse width (femto mode): ~120 fs
• Pulse width (pico mode): ~2 ps

Levante OPO

• This is a picosecond OPO pumped by a frequency doubled Nd:YVO-Laser (523 nm). The signal and idler are tuneable from 690 to 990 nm and 1150 to 2300 nm respectively.
• There is perfect spatial and temporal overlap of signal and idler beam, which can be outcoupled on one joint beam axis.

Adjacent Cell Culture Room

• Class II cell culture hood
• Incubator

Adjacent wet lab facility