Scanless two-photon microscopy with a 30 fs laser by means of a diffractive dispersion compensation module

Abstract

Summary form only given. Scanless two-photon microscopy uses a spatial light modulator (SLM) to shape the incoming laser beam into any user-defined light pattern. These microscopes, that do not contain mechanically moving parts, improve the temporal resolution of conventional scanning multiphoton microscopy since they highly mitigate the severe temporal limitations inherent to sequential scanning of the sample [1]. However, diffractive optical elements encoded into a SLM for multiphoton microscopy prevent the use of ultrafast sources (say, pulses shorter than 100 fs) due to huge dispersion (both spatial and temporal), which limits high resolution nonlinear excitation [2]. We have engineered a simple dispersion-compensated module (DCM) based on the diffractive lens-pair configuration shown in Ref. [3] that permits to extend the range of pulsed sources available for scanless multiphoton microscopes to ultrashort femtosecond pulses. In this way, we strongly alleviate some unwanted effects (specifically, spatial chirp and pulse-front tilt).Our experimental setup is shown in Fig. 1(a). A mode-locked Ti: sapphire laser that emits pulses of 30 fs temporal width, 800 nm central wavelength, at 1 kHz repetition rate is used as pulsed source. The pulsed laser beam impinges by means of a beam splitter onto a Fourier computer generated hologram (CGH) encoded into a phase-only SLM. The dashed box in Fig. 1(a) is the DCM. It is made up of a lens achromat L1 (focal length f1 300 =) mm coupled to a diffractive lens pair, DL1 and DL2. The focal lengths of DL1 and DL2, for the central wavelength of the laser are f01 = -150 mm and f02 150 = , mm respectively. The distances between the optical elements are l=300 mm and d=d'=150 mm. After the DCM, in order to properly excite the fluorescence signal in Rhodamine B (RB), we use a telescope with a refractive lens L2 (focal length f2 100 =) mm and a 20X microscope objective MO1 with focal distance 10 mm. To observe the fluorescence signal, the RB plane is imaged onto a conventional CCD sensor by means of a 50X microscope objective MO2. We place a suited filter F before the CCD camera to prevent from propagation of the infrared signal.In the experiment we encoded the outline of a bicycle on the CGH. The theoretical reconstruction is illustrated in Fig. 1(b). The average power was adjusted to 3mW to ensure wide-field fluorescence signalling. When we do not use the DCM, we realize that spatial and temporal broadening of the pulse at the sample plane makes useless the diffractive SLM, and in fact no signal is recorded. The fluorescence signal was subsequently recovered when we employed the DCM (see Fig. 1(d)). The dispersion compensation abilities of the DCM preserve the temporal width of the laser pulse at the sample plane, and consequently the fluorescence signal increases with respect to the uncompensated situation. Note that the number of emitted photons at the sample depends inversely on the pulse width. Furthermore, transverse spatial resolution for the CGH reconstruction is also maintained due to the spatial chirp compensation capacity of the module. Finally, we check that diffractiondriven fluorescence signalling is possible without the DCM, but at the expense of using an extra 2mW average power to compensate for temporal broadening, see Fig. 1(c). However, the uncompensated spatial chirp leads to a blurred signal that prevents correct hologram reconstruction and thus irradiance patterning.