Nonlinear microscopy (NLM) techniques, such as Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation (SHG), are able to overcome some of the drawbacks present on conventional Confocal Laser Scanning Microscopy (CLSM) [1
]. This is in part due to the fact that the nonlinear excitation is confined to a focused volume rather than the whole illuminated beam path as it is the case for one-photon fluorescence. Therefore photo-toxicity and out of focus photo-bleaching are considerably decreased. This confinement of light is advantageous since it allows optical sectioning of the sample, enabling the reconstruction of three dimensional (3D) models. In addition, nonlinear excitation normally relies on the use of wavelengths in the near-infrared (NIR) range. At these wavelengths, besides the fact that there is reduced photo damage, Rayleigh scattering is also decreased enabling larger penetration depths.
A key element in a nonlinear microscope is the use of an ultrafast laser. These are natural sources that are able to produce the required high intensities needed for exciting nonlinear processes. Historically, Ti:sapphire sources have been used in NLM due to its available large peak powers along with its large tunability range. However, its complexity, high price and maintenance requirements, have limited the widespread adoption of these powerful imaging techniques into daily routine biomedical applications.
Efforts in the past have explored developing compact, lower-cost, and easy to use ultrafast semiconductor saturable absorber mirrors (SESAM) for modelocking diode-pumped solid-state lasers [3
]. Compact designs can be realized by “folding” long cavities using careful mechanical design, or by increasing the repetition rate of the lasers which naturally allows short cavity lengths. However these sources have been limited by the available peak powers. More recently, compact ultrafast Cr-doped laser systems such as Cr:LiCAF, Cr:LiSAF, Cr:LiSGAF, lasers [4
] (see Ref [4
], Table. 2.1.2 for an overview on these sources) have been produced. Some of these have been employed for nonlinear TPEF imaging [7
] and although average powers of up to 500mW have been demonstrated [6
], they are often limited in their ability to sufficiently scale their average power.
Other alternative sources based on fiber lasers [12
] and semiconductor laser diodes with amplification schemes [15
] have also been successfully demonstrated as compact lasers for NLM applications. Fiber lasers can generate very short pulses via passive mode-locking, however, they are limited to wavelengths around 1030 nm and 1550 nm. In semiconductor lasers with amplification schemes, i.e., gain-switched laser source based on vertical cavity surface emitting lasers (VCSELs) [16
], gain-switched InGaAsP Distributed-Feedback-Bragg (DFB), laser diode [17
] and an external cavity mode-locked laser diode consisting of multiple quantum wells (AlGaAs) [15
], the simplicity and compactness of these systems is compromised, as they need several stages to compress and/or amplify the pulses.
One key aspect for optimizing a compact laser for TPEF or SHG microcopy is the critical trade-off between repetition rate of the laser and the multiphoton signal strength generated [18
]. The signal strength in TPEF or SHG imaging scales as the product of the peak power times the average power (assuming image spot size, absorption, sample, detection path, etc. remain constant). This two-photon figure of merit (FOM2p
) allows one to make relative comparisons of laser sources as a function of their average power, pulse duration, and repetition rate (since peak power equals average power divided by repetition rate divided by pulse dlocking howeveruration). Typical Ti:sapphire lasers on a TPEF sample give a peak-power-average-power product (FOM2p
) of approximately 1 W2
(e.g. 200 fs pulse duration, 80 MHz repetition rate, and 4 mW average power at the sample). Alternative laser sources should have a FOM2p
value of a similar order of magnitude. Higher repetition rate lasers allow for more compact designs, but require higher average power to achieve the same FOM2p
value. Repetition rates in the multi-Gigahertz range have been demonstrated in TPEF imaging, but ultimately require average powers that begin to impact sample viability.
Here we experimentally show, for the first time, that an ultrashort pulse semiconductor disk laser (SDL) or vertical extended cavity surface emitting laser (VECSEL) that is modelocked by a “quantum dot-engineered” SESAM [20
] can be in fact used for nonlinear microscopy. In fact, SDLs provide a compelling source for TPEF and SHG, as they combine key features such as excellent beam quality, output power, short pulse durations, amplitude stability, and can be made to operate at a large set of wavelengths, well-matched to key TPEF dyes, while maintaining simplicity and ease of operation. In addition, the repetition rate of these lasers can be adjusted into the 100’s of MHz to 1 GHz, resulting in a compact laser cavity, but still working in a range where the FOM2p
can be reasonable (compared to the standard Ti:sapphire laser) without requiring excessively large average powers. SDL’s also have the potential for low-cost manufacturing, as their gain element is based on wafer technology and because they have a relatively simple laser cavity design requiring only relatively low-cost and low-brightness pump diodes. Thus, in this work, we demonstrate the use of a modelocked VECSEL [20
], with a footprint of only 140x240x70 mm, for in vivo
multiphoton microscopy. The VECSEL gain chip is mode-locked with a quantum-dot SESAM [21
] to produce 1.5 ps pulses at 500 MHz with an output average power of 287 mW at 965 nm. Importantly the laser output wavelength brings the advantage that TPEF of the Green Fluorescent Protein (GFP), one of the most widely used fluorescent markers for biological applications, has its two-photon action cross section maxima around this operating wavelength [2
]. Exciting the GFP at this wavelength substantially relaxes the required FOM2p
and corresponding average and peak power values needed for TPEF-based imaging. This is demonstrated in both, fixed samples and in vivo
by imaging prepared slides containing different dyes and Caenorhabditis elegans (C. elegans)
nematodes expressing GFP in a specific set of motoneurons respectively. In addition, the extended versatility of the laser is shown by presenting SHG images of pharynx, uterus and body wall muscles, followed by a demonstration of the ability of this source to excite various commercially available dyes.
The successful implementation of this non-expensive, maintenance-free, turn-key, compact laser system in a wide range of biological applications based on different markers and SHG could potentially facilitate the wide-spread adoption of nonlinear imaging techniques for “real-life” applications.