Multiphoton Microscopy / Fiber Laser Simultaneous multi-color, multiphoton fluorophore excitation using dual-color fiber lasers Matthias Handloser, Tim Paasch-Colberg, Bernhard Wolfring TOPTICA Photonics AG Advanced microscopy techniques provide crucial insights for a deeper understanding of biological and chemical processes. Due to its nonlinear excitation character, multiphoton imaging (e.g. TPEF, SHG, THG) enables a deeper penetration depth and higher spatial resolution than conventional techniques. Here we present a novel approach to two-photon excitation at various wavelengths simultaneously using TOPTICA s FemtoFiber dichro design biomp. The presented technique employs two femtosecond laser pulses at different wavelengths to stimulate a 2-color 2-photon excitation of GFP fluorophores inside the irradiated sample. This excitation corresponds to an excitation at a third, virtual wavelength. In a similar way the laser can excite most conventional fluorophores simultaneously. Fig. 1: TOPTICA S FemtoFiber dichro biomp fiber laser provides two synchronized femtosecond laser pulses at different wavelengths to enable 2-color 2-photon excitation. Introduction Multiphoton microscopy has become a popular method for cellular imaging. The nonlinear character of the fluorophore excitation results in an intrinsic three-dimensional resolution due to the high penetration depth, combined with a higher resolution compared to conventional techniques. For an efficient excitation of fluorescent labels, a pulsed laser source is required. Titanium-sapphire (Ti:sapph) lasers are frequently used due to their relatively reliable pulse parameters and broad wavelength tuning range. Covering about 700 1000 nm with ~100 fs pulses, Ti:sapph lasers enable excitation of most important fluorophores that are used for multiphoton microscopy. Nevertheless there are several disadvantages when using Ti:sapph lasers, especially when Page 1
several fluorophores that absorb light at different wavelengths are studied in one sample. In this case it is necessary to tune the wavelength of the laser for each particular fluorophore. In addition, Ti:sapphs are relatively large and require water cooling, as well as regular maintenance. Ultrafast fiber lasers for multiphoton microscopy Femtosecond fiber lasers are an alternative solution for fluorophore excitation in multiphoton microscopy. They are compact, reliable and maintenance-free sources that require no water cooling. They operate at a fixed wavelength, therefore they are the perfect choice for experiments that do not require a wavelength tunability a precondition that is quite often fulfilled, e.g. in two-photon excitation of Alexa 488 using a wavelength of 780 nm. 2-color 2-photon absorption using TOPTICA FemtoFiber dichro biomp To address several fluorophores simultaneously at different wavelengths with fiber lasers, another adapted excitation scheme must be employed. In conventional 2-photon excitation, two photons of identical wavelength are absorbed by the sample, e.g. 2*780 nm or 2*1040 nm, c.f. Fig. 2 (a) and (c). If femtosecond laser pulses of different wavelength overlap temporally and spatially in the laser focus at the sample, a process called 2-color 2-photon excitation can be stimulated. In that case, the sample absorbs two photons of different wavelength, e.g. 780 and 1040 nm, cf. Fig. 2 (b). This excitation corresponds to a conventional 2-photon excitation at a virtual wavelength of λvirt = 890 nm corresponding to the following equation: Fig. 2: Various pathways for two-photon excitation fluorescence (TPEF). On the one hand, two identical photons can be absorbed at different wavelengths each (a) and (c), alternatively absorption of two photons at different wavelengths can stimulate 2-color TPEF (b). TOPTICA s FemtoFiber dichro biomp (cf. Fig. 1) simultaneously provides two intrinsically synchronized laser beams with sub-150 fs pulses at different wavelengths of 780 and 1040 nm out of one box, even from the same aperture. The system includes a semiautomatic time-delay calibration, as well as an adjustable spatial beam-overlap (z-focus alignment) between both laser beams to guarantee for the spatial and temporal overlap of both beams at Page 2
one sample position to access the virtual wavelength created by photons of both beams. These features allow to cover all three excitation pathways described in Fig. 2 simultaneously, i.e. exciting fluorophores at 700-1100 nm is feasible without tuning the wavelength. For example GFP, YFP and Alexa fluorophores can all be excited at the same time, with acquisition times that are comparable to single channel imaging. Excitation of GFP-Neutrophil Cells To determine the feasibility of efficient 2-color 2-photon excitation of multiple fluorophores simultaneously, a TILL Photonics Intravital2P confocal microscope has been combined with a TOPTICA FemtoFiber dichro design biomp. The obtained confocal scan map is compared to the resulting image after standard 2- photon excitation by exciting the sample material with a Ti:sapph oscillator at the respective wavelength of 890 nm, serving as reference measurement. Both laser systems share the same optical beam path to allow for proper comparison of the retrieved 2- photon scan map. All images were recorded by appropriately spectral filtering of the 2- photon induced second harmonic signal generated by the investigated fluophores used for labelling the sample material throughout the experiment. A parasagittal section of a mouse brain s hippocampus was prepared as sample, whereas about 10 percent of the neurons express YFP, and neutrophil white blood cells express GFP. The neurons in the hippocampus are pyramidal neurons, which have large cell bodies and a long protrusion, the dendrite. Additionally, there are small protrusions originating from soma and dendrite. These form a dense mesh between the layer of cell bodies and the part that contains only dendrites. Dendrites run in parallel. Neutrophils crawl between cells and dendrites. They are small, have very short protrusions, if at all, and they are less brightly stained. They are most obvious in parts of the images dominated by pyramidal neuron dendrites. Page 3
Experiment Experimental results obtained by the described experimental procedure can be seen in Figs. 3-7. By exciting the investigated sample material at 780 nm only no 2-photon excited fluorescence (TPEF) signal is observed (Fig. 3), due to the mismatch of excitation and absorption spectral region. The green protein YFP, used for labelling the material, absorbs light from 450-550 nm. Fig. 3: Microscopy signal recorded with FemtoFiber dichro design biomp at 780 nm only. No signal is observed since YFP absorbs in spectral range between 450-550 nm. By exciting the investigated the sample material at a wavelength of 1040 nm only, corresponding to a two photon excitation of 520 nm, a strong TPEF signal is observed from the YFP linked to the neurons as depicted in Fig. 4. Fig. 4: FemtoFiber dichro design biomp at 1040 nm only, leading to 2-photon excited fluorescence of the YFP neurons. Page 4
Simultanous excitation of 780 nm and 1040 nm provided by the novel FemtoFiber dichro biomp leads to a strong TPEF signal as shown in Fig. 5. By setting the relative temporal delay of both beams to zero (built in motorized and controlled delay stage) and overlapping the excitation focii spatially (built in adjustment of z- focus position) the laser systems allows to excite the sample additionally at the virtual wavelength of 890 nm. This enables a controlled excition of the GFP linked to the neutrophil blood cells as visualized in Fig. 5 and in the zoom of one region. Fig. 5: TPEF fluorescence map excited simultanously at different wavelength emitted by the FemtoFiber dichro design biomp. Both beams share the same beam path to guarantee a spatial overlap at the sample. By tuning the respective temporal delay of both beams to zero a virtual excitation at 890 nm takes place exciting also the GFP neutrophil cell as shown in the inset. A direct comparison the retrieved SHG-map after excitation directly at 890 nm (Fig. 6) indicates the efficiency of the SHG generation of the virtual wavelength emitted by the FemtoFiber dichro design biomp and serves as reference. Page 5
Fig. 6: For comparison, the same section of the sample was studied using a femtosecond Ti:sapph laser at 890 nm for fluorophore excitation, showing a similar result compared to Fig. 5. To show the efficient excitation of GFP at the virtual wavelength via 2-color 2-photon excitation using the FemtoFiber dichro design biomp, the temporal delay between both laser pulses can be altered. If the pulses do not overlap, the previously obtained GFP signal (cf. Fig. 5) vanishes, as shown in Fig. 7. Fig. 7: The FemtoFiber dichro design biomp was used with both colors, 780 and 1040 nm, as previously. In contrast to Fig. 5, the temporal delay was altered, such that the laser pulses do not overlap at the sample anymore. As a consequence, the GFP neutrophil cells vanish, whereas the YFP neurons are still visible. The efficiency of the virtual excitation by calibrated reference measurements and the respective quantum yield for different materials will be evaluated in a next step. Page 6