Femtosecond lasers for THG Microscopy

: THG image of cell division in C. elegans embryo. Courtesy of R. Loza-Alvarez & R. Aviles-Espinosa, ICFO Institute.

For various studies in different research fields, suitable techniques for long-term imaging of living cells, tissue or model organisms are required. THG microscopy is an attractive technique for this kind of applications as it yields intrinsic high resolution without the need to stain the specimen. The FemtoFiber pro is a fiber laser that greatly simplifies the integration of non-linear microscopy techniques in biological research as it is a compact, robust and inexpensive system that requires only minimum maintenance. The FemtoFiber pro IR for example emits at 1560 nm, generating a THG signal in the visible range, compatible with the integral optics of standard microscope systems and efficiently detectable with conventional PMTs. For various studies in different research fields, suitable techniques for long-term imaging of living cells, tissue or model organisms are required. THG microscopy is an attractive technique for this kind of applications as it yields intrinsic high resolution without the need to stain the specimen. The FemtoFiber pro is a fiber laser that greatly simplifies the integration of non-linear microscopy techniques in biological research as it is a compact, robust and inexpensive system that requires only minimum maintenance. The FemtoFiber pro IR for example emits at 1560 nm, generating a THG signal in the visible range, compatible with the integral optics of standard microscope systems and efficiently detectable with conventional PMTs.

Live-cell imaging. Enormous efforts are being made in order to understand the processes involved in illnesses or pathologies and for finding treatments or ways to prevent them. However, studies on single cells or isolated tissue are not able to mimic the metabolism of a complete organism as this involves a complex interplay between various tissues and multiple signaling pathways. For this reason, model organisms, like Caenorhabditis elegans (C. elegans) or Zebrafish, are used for systemic studies. In order to image the structure of living cells or even whole organisms, imaging techniques are needed that yield three-dimensional (3D) resolution, have a high penetration depth, do not alter the observed structures and – are not harmful to the imaged specimens.
Fluorescent microscopy techniques require staining of the structures with fluorescent dyes. These dyes however, may potentially alter the structures and their function or even be not compatible with live-cell imaging at all. Furthermore, for linear excitation fluorescence techniques, photobleaching and phototoxicity are issues that often hinder long-term live-cell studies.

THG-Microscopy. The THG technique is a coherent non-linear imaging technique and represents an ideal tool for live-cell studies: It provides intrinsic high resolution in three dimensions, optical sectioning capabilities and requires no labeling. In a THG process the interaction of three photons with a nonlinear material creates a new photon having three times the energy and 1/3 of wavelength of the incident light, respectively. This virtual-level transition transfers no energy to the sample, providing an imaging method without photodamage and photobleaching. By utilizing the intrinsic nonlinearity of biological samples, the samples can be imaged while the specimen and its metabolism remain unaffected.
As for other higher-order processes like 2-photon absorption or second harmonic generation (SHG), very high powers are required in order to generate a detectable signal. Therefore these nonlinear techniques make use of modelocked ultrafast lasers that emit pulses with very high peak power and duration of only femtoseconds to picoseconds. These techniques show intrinsic 3D capability with submicron resolution as only in the focus of the objective the light intensity is high enough to generate a detectable THG signal. The excitation light for all techniques is in the (near) infrared range, resulting in large penetration depths due to reduced scattering at higher wavelengths.
THG is a coherent process, meaning that the signals of adjacent molecules will interact with each other and phase matching has to be accounted for. This means, direction and polarization of the signal depend on the local distribution of the molecules in the focus. The phase matching condition normally is fulfilled in forward-direction. Therefore, most THG microscopes are constructed in a forward detection arrangement.
While second-harmonic generation (SHG) is based on non-centrosymmetric structures (limiting this technique to structures like collagen, myosin and microtubules), THG does not have any symmetry requirement. It only relies on the change of the nonlinear properties within the sample. Lipid droplets for example generate a strong THG signal and also nucleoli inside the nucleus. However, one has to keep in mind that the THG signal is sensitive to all changes of the nonlinear properties, thus providing a less specific signal compared to e.g. coherent anti-Stokes Raman scattering (CARS), which offers a high chemical selectivity. Although THG is not selective to a specific structure only, its technical realization is very simple as only a single laser is required and it is especially useful when combined with other non-invasive techniques in a multimodal approach. Here, complementary signals give in-depth insights into the examined structure.

Setup for THG microscopy. For THG microscopy typically multi-photon microscopes are used. These are laser-scanning microscopes equipped with dedicated objectives, which are corrected for excitation wavelengths in the infrared. A mode-locked ps- or fs- pulsed laser in the range between 700 nm and 1000 nm is coupled into the microscope to illuminate the sample. x/y-galvanometric mirrors scan the sample in the lateral direction and the resulting 2D image is acquired pixel wise. Z-scanning is achieved either by changing the axial position of the objective or of the microscope stage. This way images from different focal planes are obtained for 3D imaging. The THG signal can be detected in forward or backward direction by a photo multiplier tube (PMT). Most setups are constructed in a transmitted light arrangement. Here, a second objective, focusing on the same spot as the illuminating objective, detects the signal that propagates in the forward direction. In front of the detector short-pass filters block the incident laser light. Some structures, such as Hemoglobin, Melanin, Elastin or NADH, show strong autofluorescence. A narrow band-pass filter at 1/3 of the laser wavelength therefore blocks the autofluorescence signal.

TOPTICA’s added value. So far, only a limited number of THG studies have been published. One reason might be that up to now mainly large and expensive Titanium-Sapphire have been used, systems which are largely found in dedicated laser laboratories with technically skilled scientists to operate the laser. In order to make ultrafast technology also available for biology labs or clinics, TOPTICA has designed the FemtoFiber pro lasers. These are turnkey laser systems for simple hands off operation, based on alignment-free fiber technology and saturable absorber mirrors (SAM) to ensure stable pulsing. Their small (A4/letter format) footprint and easy operation greatly simplifies the integration of THG in biological research.
Another reason that the application of THG is still very limited might be the fact that the Titanium-Sapphire lasers emit in the range between 650 to 1100 nm, yielding a THG signal in the UV range (220 nm – 360 nm). For these setups dedicated UV-grade optics has to be employed. However, if an excitation wavelength above 1200 nm is used, the THG signal will be in the visible range, optimal for the integral optics of a standard microscope. TOPTICA’s FemtoFiber pro IR for example emits at a center wavelength of 1560 nm, generating a THG signal at 520 nm. Another advantage of a signal in the visible range is that it falls near the peak sensitivity region of conventional PMTs allowing very efficient signal collection. Recently the strong potential of THG at 1550 nm for high resolution 3D in-vivo imaging of C. elegans has been demonstrated [1].
The FemtoFiber pro greatly simplifies the integration of non-linear microscopy techniques in biological research. A frequency-doubled version of the same fiber laser source is available and can be used for a multimodal microscopy approach for further investigations. This way, complementary information to the THG signal can be obtained by the implementation of other techniques such as 2-photon fluorescence microscopy or SHG in the same setup.