Multiphoton microscopy

Understanding how brain works is one of the most exciting goals for the next decades. Ultra-short pulse lasers became an indispensable tool to achieve this goal and nowadays they are widely used in the so-called Multiphoton Excitation (MPE) microscopy. Compared to single photon fluorescence microscopy, MPE microscopy allows imaging of living cells at an unprecedented depth and precision. In MPE the fluorophore excitation is driven by two-three photon absorption, which is a threshold phenomenon occurring only where the optical intensity is sufficiently high. In this way, the excitation occurs only at the focal point of the laser beam leading to extremely high signal to noise ratio. Moreover, by using near infrared ultra-short pulse lasers, the scattering phenomena are strongly reduced and imaging of deep tissue layers of central nervous system is made possible.

 

In vivo two-photon fluorescence imaging of jRCamP1a-expressing neurons in the mouse neocortex at the depth of 540 µm using the LITHIUM SIX 1050 laser source (1047 nm, 140 mW on the sample). Courtesy of Dr. M. Pisoni, Dr. A. Sattin, and Dr. T. Fellin, IIT Genoa

Lithium Lasers offers an ultra-compact and robust laser with several fixed wavelengths in the near infrared region

LithiumSix1050 is a high-power laser emitting spatially and temporally clean ultra-short pulses at high repetition rate with central wavelength at 1050 nm. The majority of near infrared fluorophores used in neuroscience are efficiently activated at the wavelength of 1050 nm. However, a wavelength shifted by 20-30 nm from both sides can be convenient in dual color functional imaging experiments or when functional imaging is combined with optogenetic neuronal excitation. Our laser is now available with several fixed wavelengths in the range from 1030 to 1070 nm. The possibility to choose a preferred wavelength according to specific experimental needs enables to optimize your functional imaging experiments. This laser is also available with different repetition rate: 80 MHz for functional imaging experiments and 40 MHz for optogenetics.

 

Broadband CARS microspectroscopy

Coherent anti-Stokes Raman scattering (CARS) is a nonlinear optical microscopy technique that relies only on molecular vibrations. It provides non-invasive and label-free imaging of specific biomolecules at high-resolution. CARS microspectroscopy has been widely used for cell imaging, tissue imaging and material science. In CARS microscopy two light sources that simultaneously excite the sample are required, the first of which is referred to as the pump and the second one is the Stokes. When a broadband Stokes signal is used in combination with a narrow band pump signal an instantaneous excitation of a broad variety of vibrational modes is achieved and real time characterization of molecular structures can be acquired. This novel method called Broadband CARS (B-CARS) or Multiplex CARS can detect a full range of spectral information in real time, in contrast to other CARS techniques that can only acquire information at a certain frequency at a time. B-CARS has a good imaging speed and spatial resolution and can be used to achieve quantitative analysis.

Requisites of laser systems to generate broadband Stokes signals for B-CARS microscopy

In order to drive an efficient excitation of all vibration modes pump and Stokes signals must be well synchronized. To ensure this, the laser used as pump for the broadband Stokes signal generation must have transform limited, so as to ensure temporally end spatially overlapped longitudinal modes over the whole spectrum. An extra-requisite for efficient detection of small differential CARS signals is that the pump signals should exhibit low intensity noise at high frequencies.

Thanks to the low relative intensity noise and high peak power, Lithium Six 1050 represents a high-quality pump source for broadband spectrum generation through non-linear conversion.

Lithium Six can be used as a pump for very short LMA-PCF fibers to convert the narrow band high peak power pulse into a high-power broadband output. The low intensity noise of the input signal is preserved through the LMA-PCF fiber resulting in low-noise broadband Stokes signal, which ensures effective detection of small CARS differential signals. The multicolor pulses emitted by the system are temporally and spatially overlapped allowing a perfect synchronization of the narrowband pump and the broadband Stokes signal. Lithium Six provides an exceptional pump for the generation of  synchronized broadband Stokes signal for B-CARS microscopy.

Optical spectrum obtained with Lithium Six and a 5 cm long LMA 10 fiber 

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