| Used in: | Single molecule spectroscopy |
The setup consists of four fundamental parts.
1. We can use several lasers for fluorescence excitation covering basicall the whole visible and near IR spectral range: CW lasers (argon ion laser (458, 488, 514 nm), He-Ne laser, several diod lasers (405, 480, 640nm), CW Ti-Sapphire laser) for steady-state measurements and a pulsed Ti-Sapphire laser (Tsunami, 80 MHz repetition rate) and the same diode lasers in the pulsed mode for time-resolved measurements. Typical values of temporal and spectral pulse widths are 150 fs and 10 nm for the Ti-Spph laser. The diode lasers gives pulses of about 60 - 80 ps long. A motorized flipping mirror allows fast and easy switching between the pulsed and CW laser.
2. For direction of the excitation light to the sample and obtaining the sample image we use a home built wide-field fluorescence microscope based on a commercial Olympus IX-71 inverted microscope. Wide-field refers to the fact that the excitation laser light illuminates a “wide area” of about 30 microns of the sample.
3. To detect image of the sample light we use a 512X512 pixels CCD camera (Photometrics, Cascade 512B or Photon Max) with on-chip multiplication gain.
4. An avalanche photodiode (APD) (Micro Photon Devices, crystal size 100 m, 250 cps dark counts) is used for time-resolved fluorescence decay measurements.
A microscope with an inverted configuration gives several benefits. For instance when systems in solutions are studied (e.g. biological samples) the fluorescence can be excited and collected through bottom windows of the sample cells. Also a large sample compartment like a cryostat or a vacuum chamber can be easily fixed the microscope sample stage.
Single molecules (SM) of organic dyes or nanoparticles have small absorption cross-sections ~10-16–10-14 cm2 (depending on number of chromophores). Due to that the fluorescence intensity from a SM is also very small. Therefore even a slightest amount of background light easily drowns the SM fluorescence signal. To be able to detect and analyse SM fluorescence it is crucial to have as high as possible light collection efficiency and as low as possible unwanted background signal. The origin of the background light is scattering of the excitation light, luminescence of optical components of the setup and luminescence of the sample substrate and impurities in the sample itself. To block the excitation light of reaching the detector co-called “filter cube” is used. The filter cube contains excitation and emission filters together with a dichromatic beamsplitter also called dichroic mirror, see Figure 1.
We use a long working distance dry objective lens Olympus LUCPlanFl 40x with numerical aperture (NA) 0.6. This objective has also an adjustable correction for imaging though a glass window up to 2 mm thick. We need this in order to be able to work with samples placed in a vacuum chamber or in a cryostat.
NA determines the resolving power and the ability to collect light and it is defined by NA = nsinθ, where n is the refractive index of the medium surrounding the objective (air in our case) and θ is half the angle of the cone of the collected light, see figure 2. The ratio between the number of photons collected by the objective lens and emitted by a molecule (light collecting efficiency) is 10% in our case. To calculate the resolving power one can use the Sparrow criterion. The criterion is fulfilled if the intensity in the mid point between the objects equals to the intensity of the objects. According to Sparrow criterion the smallest resolving distance is given by d = 0.51λ/NA, which is ≈ 0.5 μm for λ=540 nm and NA = 0.6.
The total magnification of the system is approximately 80X. It originates from the magnification of the microscope itself (40X at image plane A1 (see figure 1)) and 2X magnification of the imaging lens L2. In the end we have a spatial length scale of 200 nm per pixel at the CCD camera.
The objective is used to illuminate the sample with an excitation light. If the laser beam coming to the objective lens was collimated the excitation spot at the sample plane would be as small as diffraction limited spot (ca. 0.5 μm). In order to get image we need to illuminate an area, which is much larger then the microscope resolution. To increase the illuminated area a defocusing lens (L1) is placed into the excitation beam at some distance before the objective lens. The excitation spot in a typical case has a diameter of approximately 30 μm at the sample plane (A0). To be able to attenuate the laser intensity neutral optical density filters are used.
However, even if low irradiation intensities are used (we typically work at less then 80W/cm2) a sample can get damaged due to photo-oxidation if it is not protected against oxygen. Photo-oxidation can be seen as decreasing of fluorescence intensity and complete disappearing of fluorescence after a time period as short as a few seconds. To reduce influence of oxygen we put samples into a home-built vacuum chamber (10-2 Torr) or, a cryostat (10-5 Torr). The cryostat (Janis ST-500) allows also keeping the sample at any temperatures from 10 to 300K.
In the basic setup we use a CCD camera to obtain fluorescence image of a sample. The image contains information about number of detected photons (fluorescence intensity) during the image acquisition time, which can be as short as 10ms. Special software can capture and store consecutive images (“movies”). This allows us to observe sample dynamics.
Because we are using wide-filed excitation and detection by CCD, it’s possible to see many molecules in each image. We can extract intensity as a function of time for each molecule (or any other object) by analyzing the movie.
In order to get spectrum we put a transmission diffractive grating (Thorlabs, 150 - 200 lines/mm) in front of the CCD camera. Then we still have image of our molecules in zero diffraction order and corresponding spectra in the first order. The resolution of such a simple spectrometer is about 6 nm.
O. Mirzov, R. Bloem, P. R. Hania, D. Thomsson, H. Lin, and I. G. Scheblykin, "2D polarisation single molecule imaging of multichromophoric systems with energy transfer", Small, 2009, 5, 1877
Measuring of intensity and polarization of a single molecule fluorescence excited by differently polarized excitation gives us insights to internal organization of the studied molecule and its orientation in space. Changing of the excitation light polarization state can be done using λ/2 and λ/4 waveplates, while the fluorescence polarization can be probed by a linear polarization filter (analyser) placed in front of the detector.
Let us consider a simple chromophore (e.g. an organic dye) in which the absorption and emission dipoles are oriented in the same direction. We can obtain information about orientation of the transition dipole by measuring the fluorescence polarization (fig. 3, scheme 2). For the same purpose scheme 1 can also be used where anisotropy of absorption is measured. In the last case the orientation of the chromophore dipole is given by the excitation polarisation orientation when the chromophore absorbs the most leading to maximal fluorescence intensity.
However, usually we are interested in more complex systems like polymers and multi-chromophore nanoparticles. A polymer chain fluorescence can be excited by light with different polarization state (linear, circular etc.). The fluorescence emitted by the chain can be polarized or depolarized or something in between. The degree of polarization (referred to as modulation) of the absorption will depend on the configuration of the light absorbing chromophores within the molecule i.e. its structure. The polarization degree of emission also reflects chromophores' organization, but only those, which emit fluorescence.
Even if the modulation in absorption is low the modulation in emission could be high (fluorescence is highly polarized). This is a direct evidence of energy transfer in the system. It's also possible that energy transfer results in a change not only in fluorescence polarization degree but also in the angle between maximum absorption and emission. To see such effects we use scheme 3 where both the λ/2 waveplate and the analyzer are rotated with different frequencies by step motors while the fluorescence time transient is detected.
This technique gives all information about polarization of the light absorbing and light emitting states, which can be possibly obtained in experiments with linearly polarized light. It allows finding the relative angle between those orientations that gives the highest absorption and emission. If we know the orientations of the excitation polarization and emission polarization filters in the laboratory frame we can find absolute angles. The technique also allows to probe if energy transfer takes place or not within the system.
Note, that dichroic mirrors of the microscope may distort the chosen type of excitation light polarization depending on the polarization orientation. To make corrections for these distortions we use a Berek compensator.
We use two types of polarization setups, which we call one channel (scheme 3 in figure 3 and figure 4) and two channels configuration, figure 5.
To perform polarization-sensitive experiments according to scheme 3 a λ/2 waveplate is placed into linearly polarized excitation beam and an analyzer is set in front of the CCD camera. Berek polarization compensator was used to maintain the linear polarization of the excitation light at the sample plane after it passed the dichroic mirror and filters. By rotating the λ/2 plate by a step motor the polarization angle of the excitation light (φex) was rotated in the sample plane. The analyzer angle (φem) was also rotated by another motor with a different frequency. As a result, fluorescence intensity as a function of angles φex and φem was measured.
The setup described above we call "one-channel configuration". In this configuration blinking events can't be distinguished from fast shifts in polarization orientation. To be able to distinguish these two different processes we use a two channels configuration.
Here the emission polarization is split by a rotated analyser (wire-grid polarizer) to two components. One of them is transmitted (Itrans) and the other(Iref) is reflected by the analyser. Transmitted and reflected components have polarizations parallel and perpendicular to the analyser transmission axis respectively. The transmitted light gets reflected back by a mirror and it is passing the analyser again. The surface of the mirror is placed with a small angle relative the surface of the analyzer. Thus, we get two separated images on the CCD camera containing complementary polarization information. The benefit of this configuration is that the total intensity is recorded in that sense a shift in polarization orientation makes light go from one channel to the other. Thus recorded data can be normalized to total fluorescence intensity (Itrans + Iref) and blinking events be accounted for.
Time Correlated Single Photon Counting (TCSPC) technique is used to measure fluorescence kinetics of single molecules. For time-resolved measurements we use pulsed excitation of the sample by, for example, the second harmonic of 150 fs pulse from Ti-Sapphire laser (Tsunami, 80 MHz repetition rate) with spectral maximum at 916 nm. The light of the second harmonic (spectral maximum at 458 nm) is passing through the same set of filters (“filter cube”) as 458 nm Ar-ion laser line resulting in excitation spectrum with maximum at 458 nm and ≈10 nm spectral widths.
By flipping up the motorized mirror M1 (see figure 6) the image can be obtained at the pinhole plane (A2*). By adjusting the position the image in the pinhole plane a fluorescence light from a desired part of the image can be directed through the 100 μ pinhole (see the figure). In practice we find a pixel at the CCD camera (Xpinhole, Ypinhole) which is conjugated to the pinhole. Then in order to get light from a particular molecule though the pinhole we just move the sample (using the sample stage) or the image itself (using motorized lens L2) to place the molecule of interest to the position (Xpinhole, Ypinhole) at the CCD camera. After that we flip up the mirror and can detect fluorescent photons by a fast avalanche photodiode. We use photon-counting module from Micro Photon Devices, crystal size 100 μ, 250 cps dark counts.
Pulses from APD are then counted by PicoHarp 300 (PicoQuant GmbH). The whole system operating in time-correlated single photon counting regime had a response function of about 50 ps. Time-tagged time-resolved (TTTR) mode can be used to detect absolute arrival times of all photons together with synchronization pulses from the laser in order to obtain time resolved fluorescence decay kinetics and fluorescence intensity at any given time of a fluorescence transient.
