3.2 Fluorescence Techniques for the Observation of Single Molecules
3.2.1 Wide-field Methods
In wide-field microscopy an area of the sample is illuminated by a laser beam or an arc lamp, similar to a conventional white-light microscope. Fluorescence light of the excited single molecules in this area is separated by a set of optical filters and focussed onto an array detector, which is in most cases an intensified charged coupled device (CCD) chip. The implementation of this method in the wide-field setup used in this work will be explained in detail below in Section 3.3 and Figure 3.5. This method has two major advantages: by illuminating a micron-sized area of the sample the be-haviour of many individual molecules can be observed simultaneously; in addition, modern CCD technology allows to collect up to several thousands of images at very high frame rates. Read-out rates of 30 frames per second are easily realized in full chip mode and rates up to 100 frames per second are possible by decreasing the size of the observed area on the chip.
In order to illuminate a micron-sized area on the sample, two different methods can be used. Standard wide-field microscopes, as the one used throughout this thesis, work in epi-fluorescence, i.e. the excitation beam is passed through the same objective that is used to collect the fluorescence light (see Figures 3.3a and 3.5). This arrange-ment minimizes the amount of background coming from excitation laser light, which is leaking through the optical filters in the detection pathway. A drawback of this technique is that the widened beam passes through the entire thickness of the sample and excites molecules outside the focal plane, which contribute to background fluores-cence. A possibility to overcome this problem is total internal reflection microscopy (TIRF).108, 109 This technique makes use of the fact that light is totally reflected when it hits an interface with a medium having a smaller index of refractionnunder large an-gles (from the normal to the interface). From the area of reflection an evanescent field reaches about 100nm into the lower n material and can be used to excite specifically the molecules in this thin layer. The necessary refraction index change is for exam-ple formed by the glass/water interface between the cover-slip (cover-slip: nD=1.52) and a sample in aqueous solution (water: nD=1.33). In addition to this so-called objec-tive type TIRF, which is also an epifluorescence technique, an alternaobjec-tive method, the
prism-type TIRF can be used. Here, the incident laser is guided through a glass prism, which is placed on top of the sample and the evanescent field penetrates into the sam-ple at the prism-samsam-ple interface. Fluorescence from this region is then collected us-ing a high numerical aperture objective placed below the sample. However, the TIRF technique is restricted to probes with a strong refractive index change between the objective/cover-slip or the prism and the sample, as it is the case for many biologi-cal samples in aqueous buffer solution. It is not suited for the kind of measurements presented in this work because very little refractive index change occurs between the thin silica films with the incorporated dye molecules and the cover-slips on which the films are spin-coated. However, the problem of out-of-focus background addressed by TIRF does not arise for the thin films investigated here because they are thinner than the focal depth of the microscope (>1µm). Therefore no out-of-focus molecules can be excited by the incident laser beam and contribute to background fluorescence.
For all wide-field techniques the detected signals of the single molecules are diffraction-limited. In each frame single molecules show up as bright spots on a dark background.
Since they are much smaller than the wavelengthλ, the single molecules can be con-sidered as point like emitters, and the response of the imaging system consisting of the objective and the lenses in the detection pathway is described by the point spread function (PSF). The radial intensity distribution is described by a first order bessel func-tion, which can be fitted to good approximation by a 2D Gaussian function.110 Using a high numerical aperture (N.A.) objective and excitation light in the red part of the visible spectrum the single molecules will be imaged as spots of ca.300nm diameter (full width at half maximum) on the detector. However, the positions of these spots can be determined with a much higher accuracy by fitting a suitable peak function such as a 2D gaussian to the signal. The accuracy of the fit is determined only by the signal-to-background and the signal-to-noise ratio. If a CCD detector with a chip consisting of a pixel array is used, the magnification and thus the number of pixels on which the single-molecule signal is imaged has to be taken into account for the accuracy of the fit.
Before tackling this question in Section 3.4 a brief overview of modern CCD technology is given in the following section.
CCD Technology for single-molecule microscopy
The area detector represents the heart of every wide-field imaging setup. Its read out rate and the gain and noise of the amplifier strongly influence the quality of the data.
Therefore this paragraph gives a short introduction into the mode of operation of CCD cameras that are used for single-molecule microscopy.
A CCD detector consists of an array of silicon diode photosensors, the pixels (= picture elements), which is coupled to a charge storage region that is, in turn, connected to an amplifier that reads out the quantity of accumulated charge. Incident photons with energy larger than the semiconductor band gap create an electron-hole pair and thus an electronic charge. The quantum efficiency and spectral response of the detector is thus defined by the transmission and absorption properties of the silicon. The minimal time Timage between two images and thus the temporal resolution of the measurement is determined by the exposure timeTexp and the readout timeTreadout of the chip. The inverse ofTimage is denoted the frame rate FR (typically given in units of fps = frames per second).
Timage =Texp+Treadout =FR−1 (3.2.1)
The readout timeTreadoutof one complete image depends on instrumental parameters, like the time needed to read out a single pixel and the complete number of pixels per image. Modern CCD cameras can work in frame transfer mode to decrease the readout time. In this mode only half of the pixels on the chip are exposed during Texp (Image Section), the other half is shielded from light (Store Section), see Figure 3.4a. AfterTexp the charges from the image section are transferred to the store section, so that the image section can be again exposed while the charges from the store section are read out line by line in the readout register. In that way,Treadoutis reduced to the shift time from the image to the store section as long asTexp exceeds the time needed to readout the store section. The readout time can be decreased further by using subregions of the full chip.
For most cameras the shifting of the lines into the readout register limitsTreadout. Thus a decrease of the height of the illuminated region can improve the frame rate, whereas a decrease in the width will not provide any improvement.
In general the sensitivity of a camera is governed by its readout noise, as signals below the readout noise cannot be detected regardless of the subsequent amplifying stages.
Therefore, to gather comparably weak signals, like single-molecule fluorescence, ad-ditional on-chip signal amplification is needed. Different technologies exist to per-form this, like intensified (ICCD) or electron multiplying CCD cameras (EMCCD). Both types have been used for the measurements in this work. The structure of an EMCCD is essentially the same as of a conventional CCD, with an additional multiplication register after the serial readout register. Thus the electronic signal is amplified prior to being read at the output node, and hence the sensitivity of the device is increased.
Alternatively, the photonic signal can be increased before the CCD chip through a microchannel plate (MCP). This MCP is similar to a photomultiplier tube. The inci-dent photons release electrons from a photocathode in front of the MCP. The electrons
Figure 3.4: Different CCD structures.(a) EMCCD (electron multiplying). (b) ICCD (Intensi-fied). Adapted from111.
are then accelerated through the multiplier, consisting of a series of angled tubes, like sketched in Figure 3.4b. In the accelerating field they gain sufficient energy to knock off additional electrons along the tubes of the MCP. The multiplied electrons are then ei-ther detected directly by a special electron bombardment CCD or indirectly by using a fluorescent screen coupled with an optical fibre array to a conventional CCD. By using a pulsed gate voltage, an ICCD can achieve very short exposure times. However, for single-molecule experiments EMCCD cameras are better suited, because they provide a higher sensitivity than ICCD devices and sufficiently high frame rates.
The quantum efficiency of a CCD can be increased by exposing the sensitive region of the chip, its ’back’-side. In conventional CCD cameras the incoming light has to pass first the region with the gate electrodes attached to the ’front’-side of the chip that are used to shift the charge through the CCD. In a back-illuminated CCD the bulk silicon substrate of the chip has been thinned by etching until it is transparent and therefore the chip can be exposed at the substrate side. Through this quite expensive process the quantum efficiency can be as high as 90% for light in the visible region.