Determination of two-photon excitation and emission spectra of fluorescent molecules in single living cells Valerică Raicu*a,b, Anurag Chaturvedia, Michael Stonemana, Giorgi Petrova, Russell Funga, Dilano Saldina, Devin Gillmana a Department of Physics, University of Wisconsin, PO Box 313, Milwaukee, WI 53211-0413, USA; b Department of Biological Sciences, University of Wisconsin, Milwaukee, WI, USA ABSTRACT Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative Förster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup, represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo. Keywords: Two-photon microscope, pulse shaper, spatial light modulator, two-photon excitation spectra, two-photon emission spectra, Förster Resonance Energy Transfer (FRET).
1. INTRODUCTION With the advent of technologies for isolation, characterization, cloning and mutation of naturally occurring fluorescent proteins (such as the Green Fluorescent Protein, GFP, and its variants) that can be fused to proteins of interest in vivo, it has become advantageous to study protein distributions and interactions at their normal locations in living cells based on detection of light from fluorescent tags1-3. A breakthrough in the development of imaging techniques has been the introduction of the two-photon laser scanning microscope (TPM) by the W. W. Webb group in early nineteen nineties4-6. This microscope exploits the ability of fluorescent molecules to absorb at once two photons6 having together the necessary energy to induce an electronic excitation of the molecule (equivalent to the excitation of a single, more energetic photon). The two photons do not have to present equal energies (or wavelengths); their wavelengths only have to obey the relationship: 1/λabs = 1/λ1 + 1/λ2. In addition, this effect is possible in principle for an arbitrary number of photons. The conditions for multiphoton process to occur are created experimentally by tightly focusing the beam of a pulsed (sub-picosecond) laser through a high numerical aperture objective and thus confining the photons spatially and temporally. Because the intensity of the light is only high enough for the two-photon excitation process to occur in a small volume around the focal point, image sectioning without confocal pinhole is achieved. The main advantages of multiphoton imaging are: (1) the number of photons arriving at the detector is larger than when using a detection pinhole because image sectioning occurs without a need to use confocal pinholes that attenuate the signal; (2) any photodestruction (i.e., photobleaching) of the sample by the exciting beam is confined to the sample layer being imaged, leaving intact the rest of the sample for further study; (3) biological materials are more transparent in longer wavelengths and thus images can be obtained from deeper layers of biological tissues; (4) using two near-IR photons to excite molecules with excitation in the UV range reduces the damage to the rest of the sample that would otherwise be induced by UV light; (5) large separation between excitation (infrared) and emission (visible) wavelengths in the TPM allows for filtering out of any excitation light leaking into the detection channel, thereby decreasing the background noise and
*
[email protected]; phone 1 414 229-4969; fax 1 414 229-5589; http://www.uwm.edu/Dept/Physics/Raicu/ Multiphoton Microscopy in the Biomedical Sciences VIII, edited by Ammasi Periasamy, Peter T. C. So, Proc. of SPIE Vol. 6860, 686018, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.768306
Proc. of SPIE Vol. 6860 686018-1 2008 SPIE Digital Library -- Subscriber Archive Copy
allowing detection of signal at low expression levels; (6) finally, the absence of a confocal pinhole allows the use of nondescanned mode of detection in conjunction with CCD cameras7,8, which leads to higher detection speed. Currently, the most suitable laser for two photon microscope (TPM)6 is the modelocked Ti:Sapphire laser introduced by Sibbet et al in 19909, which provides near pulsed light with typical pulse durations of the order of 10-100 femtoseconds (10-14 – 10-13 s) and extremely high peak powers (of the order of many kilowatts). With its tunable near IR light (7001000 nm) and/or a very broad bandwidth (inherent in the short pulses) of 40 nm to 100 nm, the modelocked Ti:Sapphire laser is particularly suitable for exciting fluorescent dyes with excitation maxima in the near UV to the green part of the spectrum, as for example variants of the green fluorescent protein. Many biophysical applications, including quantitative Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), or photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. Such experiments demand very rapid wavelength tuning, which is not currently achievable with tunable Ti:Sapphire laser. In this paper we show how a pulse shaper using a computer-controlled spatial modulator can be used to rapidly tune the excitation wavelength in a TPM that already incorporates spectral resolution in the emission channel. This instrument permits acquisition of two-photon excitation and emission spectra of fluorescent molecules in single living cells.
2. DESCRIPTION OF THE TWO-PHTON MICROSCOPE 2.1 TPM with spectral resolution in the emission channel A detailed description of the two-photon microscope developed in our lab, which presents spectral resolution only in the emission channel, has been provided elsewhere8, and it will only be presented briefly herein (Fig. 1). A solid state laser (VerdiTM, Coherent Inc., CA, 532 nm) is used to pump a modelocked Ti:Sapphire laser (KM Labs, CO), which generates broad bandwidth femtosecond light pulses (
