Supplementary Table 1. Components of an FCS setup (1PE and 2PE)

Transcription

1 Supplementary Table 1. Components of an FCS setup (1PE and 2PE) Component and function Laser source Excitation of fluorophores Microscope with xy-translation stage mounted on vibration isolated optical table Adjustment and positioning of sample and light path control High-NA water immersion objective lens Laser excitation and fluorescence collection Dichroic mirrors and emission filters A) Spectral separation of laser excitation and fluorescence emission light (one dichroic mirror, one emission filter) Alternate option: B) Spectral separation for dualcolor applications (two dichroic mirror, two emission filters) High sensitivity photodetector: single photon counting module Digital single photon detection of fluorescence light Comments and details For 1PE: Continuous wave laser: Helium-Neon (543, 594, 633 nm), Argon- Ion (488, 515 nm) or solid state laser (most common 515 nm) Pulsed laser: Tunable Titanium:Sapphire laser (Coherent, Spectra Physics) with high repetition rates of MHz and short pulses of fs In vitro and in cell culture: Inverted microscope (Zeiss, Olympus) including (custom made) FCS detection unit mounted for example, on a camera port. The FCS rig must be mounted on a vibration isolated platform (Newport, Melles Griot) including the laser source (unless it is fiber coupled). In vivo: Comparable FCS setup on an upright microscope For in vitro and intracellular measurements on an inverted microscope: Coverslip corrected objective with adjustable correction collar (Zeiss, Olympus) For intracellular measurements on an upright microscope: dipping lens (no cover glass correction). IR/VIS objective lens coating with high transmission for wavelength range nm Oil immersion objective lenses are generally not recommended for FCS due to their critical distortions of the PSF for increasing focus distances from the glass coverslip. For 1PE: Standard dichroic mirrors and fluorescence filters (Chroma, AHF Analysentechnik, Semrock) depend on (A) laser excitation wavelength and (B) the fluorophore s emission spectra to be separated. 2PE dichroic mirrors and high-quality fluorescence filters should be used with maximal blocking in the IR excitation wavelength range. Specific mirrors/filters reflection/transmission characteristics depend on (A) laser excitation wavelength range and (B) the fluorophore s emission spectra. Silicon Avalanche Photodiode (APD) based single photon counting modules (Perkin Elmer) are commonly used due to their high quantum efficiency in the visible and NIR range. Photomultiplier tube (PMT) based photon counting heads (Hamamatsu) are also suitable for FCS although they provide slightly lower quantum efficiency in the visible red wavelength range and often show a higher afterpulsing effect. Detectors can be either directly coupled or fiber coupled if a more compact detection unit is preferred.

2 Pinhole Axial confinement of confocal volume Hardware correlator FCS data acquisition and processing Computer Correlator and data acquisition control and display For 1PE: A thin high quality metal pinhole (Linos Photonics GmbH, Newport) should be used. The optimal diameter for ideal FCS performance is 50 µm for a 488 nm excitation laser (and scales linearly with wavelength). Alternatively, in a fiber-coupled setup a multi-mode patch fiber (Thorlabs) with a similar core diameter can also serve as a pinhole. No pinhole is required. Nonlinear optical sectioning induces a pinhole effect comparable to use of a pinhole size of ~25 µm with 1PE. For real time FCS the most common correlators are multiple tau digital correlators (ALV Langen GmbH, correlator.com) with one TTL pulse input (and at least two for dual-color applications). No special requirements most correlator devices will operate either via a standard EPP, PCI or USB interface.

3 SUPPLEMENTARY METHODS PROCEDURE More detailed descriptions and instrument-specific guidelines for particular protocol steps are elaborated here. Step numbers correspond to those in the Procedure. Setup adjustment 3 For 1PE overfilling the back aperture [such as in the commercially available instrument, the ConfoCor2 (Zeiss)] creates a smaller, however, more distorted volume element which can be nevertheless advantageous for work in cells due to reduced collection of background light and higher brightness values. 5 For a detailed pinhole adjustment protocol for the ConfoCor2 (Zeiss), refer to Step 6 in ref 1. Calibration 9 Typical data acquisition times for in vitro measurements are usually 100 s (either 1 x 100 s or preferably 10 x 10 s measurements). The key curve fitting parameters (diffusion time and structure parameter) derived from the autocorrelation curves of the calibration dye solution can be used to define the size and proportion of the detection volume for the experimental setup at the wavelength range observed. This is important for later quantification of the experimental parameters to be studied (concentration and mobility rates) of molecules in living cells. FCS curve fitting of the resulting correlation function ideally reflects the expected brightness and diffusion time of the standard dye species as well as the structure parameter. For 1PE, underfilled back apertures will usually result in a lower S, while overfilled back apertures lead to more severe distortions of the illumination profile and a higher S. Note also that for low values of S (S < 2), the shape of the FCS decay changes significantly with S. For higher values (6 < S < ), the shape of the curve remains almost the same. 11 Alternatively, the 2PE focal volume can also be calculated if the numerical aperture (NA) of the objective lens and the excitation wavelength are known (see Eq. 1 and 2 in ref. 2) when assuming diffraction limited optics (overfilled back aperture) and using a 3D Gaussian approximation. If this approach is used, experimental confirmation of the focal volume should be done to confirm the validity of the calculated value since experimental setups are often unable to reach this idealized, calculated value. 12 For 2PE the diffusion equation in Step 11 should be modified to τ diff = r o 2 /8D to introduce a 2PE convolution factor to account for the spatial distribution being described as a squared instead of a simple Gaussian 3. However, when using calculated 2PE volume dimensions, which is defined as a simple Gaussian from the start, the diffusion equation remains unchanged as given in Step 11. Delivery of fluorescent molecules to cells 13 The fluorescently labeled molecule of interest can be delivered to the target cells using transfection, electroporation, microinjection, or lipid-mediated delivery. Since FCS requires low nanomolar concentrations of fluorescent probe, particular care must be taken to control intracellular concentrations.

4 Optimizing intracellular FCS measurements 15 Autofluorescence is a potential obstacle for FCS in cells and depends both on cell type and cell culture conditions. Therefore, after measurement conditions for a particular fluorescent probe and cell type are optimized, careful negative controls of unloaded cells are essential to check for autofluorescence (typical concentrations and count rates) in unloaded cells and characterize the behavior (correlated or non-correlated, stable or nonstable). In practice, cells will always produce some background counts with some cell types showing an initial transient autofluorescent species that will sharply decrease and become stable within seconds. This is most likely due to the fast bleaching of immobile molecules in the stationary FCS focus. Two-photon excitation (2PE) is particularly advantageous for intracellular applications since the TP cross-sections for intrinsic autofluorescence are typically low and no out-of-focus autofluorescence is generated (see below). However, autofluorescence will still not be zero under these circumstances and must be taken into consideration. For example, PC12 cells have strong autofluorescence, probably due to high concentrations of catecholamines. This renders them not ideal for FCS experiments. In contrast, HEK293 cells show very little autofluorescence under all typical conditions for both 1PE and 2PE FCS experiments. Hippocampal neurons are critically sensitive to both cell culture and experimental conditions. Immature hippocampal neurons excited at 870 nm and cultured in media containing phenol red show bright fibrous-looking autofluorescence that produces robust autocorrelation curves as seen in Fig. 2b. When cultured in phenol red-free media and excited at wavelengths of 900 nm and higher, autofluorescence is no longer an issue Analysis of intracellular FCS measurements 21 More recently, alternative fitting routines for FCS analysis have been explored and implemented, such as MEMFCS 4 (Maximum Entropy Method based fitting routine) and PGSL 5 (Probablistic Global Search Lausanne).

5 TWO-PHOTON FCS Two-photon FCS confers many benefits for intracellular FCS measurements. Here we explain the application of 2PE to FCS and the advantages of 2PE FCS. An additional protocol for determining the optimal wavelength of excitation is provided. Use of Two-Photon Excitation with FCS Besides conventional confocal detection FCS can also be accomplished in a two-photon excitation (2PE) setup, where the two-photon focus optically defines a sub-femtoliter volume and allows for efficient wide-field detection in a pinhole-free setup. 2PE requires the nearsimultaneous absorption of two low-energy photons (typically in the NIR), which use their combined energy to excite a fluorescent molecule without changing its emission spectrum. Since the TP excitation probability is proportional to the square of the intensity, the focal volume for 2PE is restricted to the area with the highest photon density. As a result, there is inherent 3D resolution of the effective observation volume. Advantages of Two-Photon FCS 2PE has been shown to provide the following advantages over 1PE, particularly for its application in cells: pinhole-free detection minimization of cumulative photobleaching reduction of intrinsic cellular autofluorescence elimination of out-of-focus background increased cell viability due to decreased photodamage, phototoxicity and photooxidation of cellular material deeper penetration depths for thick tissue due to IR excitation Particularly for intracellular FCS applications, decreased concentration depletion due to reduced photobleaching along with suppression of autofluorescence obtained with 2PE allows for longer averaging times and makes it the preferred choice under most circumstances for work in cells. However, some intracellular experiments, such as membrane work, do not suffer from the same problems (i.e. cumulative bleaching, autofluorescence) and can be effectively accomplished using 1PE FCS. Besides the use of cheaper lasers, the major advantages of 1PE FCS are the greater variety of fluorophores and, in general, higher molecular brightness values. Determination of Optimal Excitation Wavelength Ranges for Intracellular 2PE FCS Experiments For 2PE FCS the excitation wavelength can be chosen by experimentally determining the optimal region (Option A) or by checking the two-photon cross-section (Option B). In general for intracellular measurements wavelengths above 900 nm are preferred to avoid cellular damage and autofluorescence problems. Option A: The suitable wavelength region for two-photon excitation can be found by scanning with a tunable laser between typically 800 and 1000 nm. The characteristic molecular brightness values η (Hz/molecule) at fluorescence saturation are measured with FCS. During the scan, the laser intensity should be adjusted to the respective saturation level for maximal efficiency while the pulse width is kept constant at 100 fs.

6 Option B: If the two-photon cross-section is known for the particular dye of interest, a region of wavelengths for maximally exciting the fluorophore can be deduced. For example, the intrinsically fluorescent protein, egfp (enhanced green fluorescent protein) has a large absorption cross-section and can be effectively excited for 2PE FCS measurements from nm, making it well suited for intracellular FCS measurements.

7 REFERENCES 1. Bacia, K. & Schwille, P. Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat. Protoc. (in press). 2. Zipfel, W.R. & Webb, W.W. In vivo diffusion measurements using multiphoton excitation fluorescence photobleaching recovery and fluorescence correlation spectroscopy in Methods in Cellular Imaging. (Periasamy, A. ed.) (Oxford, New York, 2001). 3. Schwille, P., Haupts, U., Maiti, S. & Webb, W.W. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77, (1999). 4. Sengupta, P., Garai, K., Balaji, J., Periasamy, N., & Maiti, S. Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. Biophys. J. 84, (2003). 5. Rao, R., Langoju, R., Gösch, M., Rigler, P., Serov, A., & Lasser T. Stochastic approach to data analysis in fluorescence correlation spectroscopy, J. Phys. Chem. A 110, (2006).