While traditional solid state lasers (e.g. Ti:Sapphire lasers) have been used to demonstrate this type of application in laboratory experiments, the laser’s size and restricted operating conditions prevent the technology from being used in clinical settings where it can benefit the general patient population. IMRA’s robust, reliable femtosecond fiber lasers enable ultrafast laser techniques to be used in practical settings.
Several unique imaging and spectroscopy methodologies are possible because of the short pulse duration and the high intensity of light available from femtosecond lasers. IMRA’s compact, rugged Femtolite F/G/H and Femtolite Ultra products allow these techniques to be used in clinical and industrial environments.
Various types of imaging and spectroscopy are discussed below.
Multiphoton Fluorescence Microscopy
In a multiphoton fluorescence process, two or more photons are simultaneously absorbed by a molecule, which then emits a single photon at a different wavelength as it relaxes back to its electronic ground state. A femtosecond laser is required because the probability of simultaneous absorption depends strongly on the intensity of the light. Sufficiently high intensity is only generated at the laser focus so that 3-dimensional mapping is possible without the use of a pin-hole or aperture, as with confocal microscopy. This leads to higher resolution and the ability to identify specific features with greater accuracy than single-photon confocal imaging. The ultrashort pulse duration means high peak power levels can be achieved at the sample while maintaining low average power. The low average power reduces cell toxicity and heat-related effects in the sample.
Second Harmonic Generation / Third Harmonic Generation Imaging
In second harmonic generation (SHG) imaging, incident light is frequency doubled by the tissue structure it passes through. Third harmonic generation (THG) works by a similar process where the laser light frequency is tripled. This technique is less wavelength dependent than multiphoton excitation microscopy. Additionally, second and third harmonic generation microscopy techniques offer reduced phototoxicity compared to fluorescence microscopy. They offer endogenous contrast without the need for potentially disruptive molecular labels, and the lack of electronic absorption reduces the chance of cellular photodamage, making them attractive options for biological imaging. An example of a common use for SHG imaging is studying collagen structure.
Terahertz Imaging and Spectroscopy
Terahertz (THz) radiation lies between the microwave and infrared regions of the electromagnetic spectrum. Most dielectric materials, such as plastics, linen, and wood, are transparent to THz radiation, allowing it to “see” through obstructions in its path. THz waves have low photon energy and thus cannot lead to photoionization in biological tissues. At THz frequencies, many molecules exhibit strong absorption and dispersion rates, making THz wave fingerprinting possible. With a balance between spatial resolution and scattering, THz wave imaging provides high contrast in nondestructive inspections. A THz wave imaging modality would produce images with “component contrast” enabling THz waves to be used for diagnosis of disease, detection of pollutants, sensing of biological and chemical agents, or locating threatening objects.
Broadband THz pulses are generated by the excitation of different materials with ultrashort laser pulses, through a variety of different mechanisms including photo-carrier acceleration, nonlinear optical process (optical rectification), and plasma oscillations. The detection technologies, such as electro-optical sampling and optical switching, are usually reciprocal processes of the THz pulse generation. Time-gating by the same ultrashort laser pulses directly maps the transient electric field of the THz wave, obtaining not only amplitude but also phase information. This gives access to absorption and dispersion spectroscopy simultaneously.
The nanoJoule pulse energies, excellent reliability, and low noise in the kHz to MHz frequency range provided by IMRA’s Femtolite F / G / H femtosecond fiber lasers make them suitable sources for most THz applications. The compact size and rugged design enable these lasers to be used in even the most demanding field and industrial environments.
Fluorescent Protein Imaging
Using fluorescent proteins as labels, fluorescent protein imaging enables a broad range of experimental observations in cells and tissues. The emission of these proteins—GFP, BFP, YFP, RFP, and others—can help determine the location and dynamics of genes, molecules, and proteins. Excitation with a femtosecond laser results in more precise imaging at greater tissue depth with less interference from autofluorescence. The ultrafast pulses of a femtosecond laser also minimize photo-bleaching of the proteins and photo-damage of the tissue; this can lead to more development of in-vivo imaging and long-time imaging.
Picture & movie provided by: Prof. Matsuzaki at University of Tokyo, Japan
Sample: Mouse brain neuron cell
Indicator: Red Ca2 + fluorescent protein (R-CaMP1.07)
Laser: IMRA Femtolite FD, D-FD-1000S
Lens: x25 Objective lens (Olympus XLPLN25XWMP2)