The instruments and methods used for SMS have be extensively described and reviewed elsewhere;63
here, we summarize a few of the basic techniques.
SMS experiments generally use inverted optical fluorescence microscopes, configured in either wide-field illumination or confocal (see ). The simplest wide-field method is epifluorescence, in which an expanded excitation beam is focused at the back focal plane of the objective, producing a collimated illumination beam at the sample. Fluorescence is collected through the same objective, filtered from any scattered excitation light using a dichroic mirror and long-pass or bandpass filters, and imaged onto a camera.
Generalized microscope configurations for single-molecule imaging in living cells.
Because epifluorescence excites a large volume of sample, background signal from out-of-focus emitters can interfere in imaging thick samples. Total-internal-reflection fluorescence (TIRF) imaging solves this problem by exciting only a thin slice of the sample nearest the coverslip. Excitation light is directed into the objective off center, causing the beam to totally internally reflect at the coverslip, subsequently producing a quickly decaying evanescent field up from the surface. The fluorescence excited by the evanescent field is collected through the objective, filtered, and imaged using a camera. Because the evanescent field falls off exponentially within ~100 nm, TIRF is useful when the region of interest is very near the coverslip. A variation called quasi-TIRF (also referred to as “pseudo-” or “leaky-TIRF”) also sends in the excitation beam off-center, but not far enough for total-internal reflection; instead, a highly angled beam exits the objective and illuminates the sample in a slice thicker than TIRF but thinner than epifluorescence.
Another method to reduce out-of-focus fluorescence is confocal microscopy, which is a point-detection, scanning technique. A collimated excitation beam that slightly overfills the back aperture of the objective is directed into the microscope, producing a diffraction-limited spot at the sample. The confocal spot is scanned across the sample and emission is collected through the objective, filtered, focused through a pinhole (which rejects out-of-focus light), recollimated, then focused onto a point detector. Confocal imaging is not constrained to be near the coverslip, so can be used to image deeper into a sample or for three-dimensional scanning. The primary drawback of confocal is that it requires scanning the stage or the beam and a point detector, so multiple parts of the sample cannot be imaged simultaneously. This makes widefield configurations much more practical for single-particle tracking and imaging dynamic structures.
In order to measure the stream of photons from a single emitter, a detector must exhibit low dark counts, high quantum efficiencies over a range of wavelengths, and low noise (from readout, electron multiplication, or analog-to-digital conversion). For details regarding detector types, characteristics and capabilities of different detectors, quantitative resolution and sensitivity parameters, sufficient signal-to-noise ratios for SMS detection, and other technical details see references 63, 64
There are two classes of detectors for SMS, single-element or point detectors, and two-dimensional array detectors. Point detectors for confocal imaging include photomultiplier tubes (PMT), avalanche photodiodes (APD or SPAD), or hybrids thereof. While PMTs have large detection area (~1 cm2) and ps–ns time resolution, APDs have higher quantum efficiencies and more easily detect single photons; moreover, APDs have very low dark counts, have faster time resolution, and output a digital signal that can easily be interfaced with a computer. The major drawbacks of APDs are (a) the small detection area, which makes aligning onto the sensor more difficult, and (b) the limited photon detection rates.
Wide-field imaging configurations allow the use of multidetector arrays or cameras, such as charge-coupled devices (CCD). Modern Si CCDs often include on-chip electron multiplication to increase sensitivity and reduce readout noise; moreover, frame-transfer technology permits faster imaging rates by performing the slow readout step on a separate, dark section of the chip. These cameras typically have quantum efficiencies >80% for the visible spectrum and frame-integration times of 10–100 ms, or faster for fewer pixels.
High quality lenses, mirrors, and filters are especially important for the ultrasensitive detection required for SMS. Immersion objectives with high numerical aperture (NA ~1.4) are necessary to collect as much of the emission as possible, but can complicate polarization. In addition, objectives for SMS should be achromatic and corrected for the coverslip material and thickness. Objectives specifically designed for TIRF are carefully designed to allow the excitation beam to be far off center.
Optical filters and dichroic mirrors must not fluoresce, must pass as much fluorescence as possible, and must reject all pump scattering and as much spurious signal as possible. Thus, long-pass filters must have sharp cut-on spectra, with optical density 6+ at shorter wavelengths. Bandpass filters can be helpful to remove longer-wavelength background fluorescence or excess Raman scattering from water, but it is important to overlay the filter's transmission spectrum with the emission spectrum of the fluorescent label to avoid rejecting too much of the emission. Filters and lenses inside the microscope should be anti-reflection coated and aberration-corrected. For instance, thin dichroics can easily bend and distort the image; 2-mm or thicker dichroics are preferable.
Light sources for SMS are many, but are usually lasers. Single-frequency dye lasers were used in the early cryogenic SMS experiments, where finding the narrow absorbance peaks required tuning the excitation light. At room temperature, gas (e.g. argon-ion, helium-neon, etc.), diode, or solid-state lasers are typically used. While lasers are necessary for some techniques, epifluorescence excitation is possible using broadband sources such as arc lamps or light-emitting diodes. White-light or fiber lasers can also provide broadband light, produced by nonlinear optical effects when high-energy pulses (from a titanium-sapphire or other pulsed laser) are transmitted through special optical fibers. Broadband sources can be convenient because they are tunable to a range of colors, but are more difficult to filter than a monochromatic excitation source. In all cases, the excitation source should be filtered to reject unwanted leakage of the other colors or laser lines.