Journal of Microscopy, Vol. 235, Pt 2 2009, pp. 119–123 Received 24 January 2009; accepted 31 March 2009 A simple twist for signal enhancement in non-linear optical microscopy A.K. DE & D. GOSWAMI Department of Chemistry, Indian Institute of Technology Kanpur, UP-208016, India Key words. Cellular imaging, continuous wave and pulsed excitation, fluorescence enhancement, laser-scanning microscopy, two-photon absorption. Summary We describe a very simple but elegant approach to two-photon fluorescence signal enhancement by intensity modulation with immediate application in two-photon laser-scanning fluorescence microscopy. This method of enhancement shows potential application in any microscopic technique that result from non-linear photon absorption and plays a pivotal role in live cell imaging. Introduction One of the state of the art technologies of modern day optical imaging, laser-scanning two-photon fluorescence microscopy (Denk et al., 1990) is based on two-photon absorption (TPA) predicted back in 1931 (Goeppert-Mayer, 1931) and experimentally first demonstrated in 1961 (Kaiser & Garrett, 1961) shortly after the advent of lasers. TPA is a third- order non-linear optical phenomenon where two photons are absorbed simultaneously and the probability of absorption varies as the second power of the intensity (I ) of incident radiation (Boyd, 1992). In practice continuous-wave (cw) lasers are not used for non-linear optical microscopy as very high intensity of the incident radiation is required at the sample. This is taken care of by using high-repetition rate lasers producing ultrafast pulses with very high peak power at moderate time-averaged power. Now, the typical time lapse between the pulses of a high-repetition rate laser is nearly 10 ns. On the other hand, in a laser-scanning non-linear optical microscope the images are constructed in pixel by pixel manner and the average laser dwell time per pixel is 10 μs. Therefore, each pixel is illuminated by a bunch of ∼1000 ultra-short pulses and we can consider the illumination to be a quasi continuous one (Denk et al., 2006). Correspondence to: D. Goswami. Tel: +91-512-259 7187; fax: +91-512-259 7554; e-mail: dgoswami@iitk.ac.in In this paper, we discuss the huge signal enhancement in two-photon fluorescence laser scanning microscopy by intensity modulation of a train of ultrafast laser pulses. Based on our earlier work (De & Goswami, 2009a), we demonstrate how this technique leads to significant fluorescence enhancement with reduced photo damage. Materials and Methods In our experiment, we used ∼100 fs pulsed excitation centred on 780 nm from a mode-locked Ti:saph laser (Mira900-F pumped by 532 nm excitation from Verdi5, Coherent, Inc., Portland, OR, U.S.A.) having 76 MHz pulse repetition rate. A neutral density filter wheel was used to control the laser power. We passed the beam through an electro-optic amplitude modulator (4101, driven by a fixed frequency driver, 3363, New Focus, San Jose, CA, U.S.A.) operating at 1 MHz. When a Glan–Taylor prism-polarizer (analyser) is kept immediately after the amplitude modulator, the output signal intensity (I out ) is modulated as: I out = I in sin 2 π 2 V in V π where the suffix ‘in’ stands for inputs and V’s are the voltages with V π 19 V when operating at 633 nm. Figure 1 shows the oscilloscope trace of the output of the driver operated at maximum modulation depth. For chopping at 1 kHz, we replaced the electro-optic modulator a mechanical chopper (MC1000A, Thorlabs, Inc., Newton, NJ, U.S.A.). Finally we sent the laser beam to the scan-head of the multiphoton- ready confocal microscope system (FV300 coupled with IX71, Olympus, Inc., Tokyo, Japan) which focuses the beam onto the sample by a high numerical aperture (NA) oil-immersion objective (60×, 1.42 NA). Each of the images comprised 512 × 512 pixels and was acquired by a single scan (with a scan speed of three frames per second) using fluoview software. Images of bovine pulmonary artery endothelial C  2009 The Authors Journal compilation C  2009 The Royal Microscopical Society