Single-molecule spectroscopy (SMS) provides information on the distribution of microstates and transitions of a wide variety of biomolecules. Fundamental insights into molecular biophysics have been obtained through this suite of techniques.1
However, technical limitations prevent application of SMS to many types of processes. Particularly challenging are processes that involve (1) weak intermolecular interactions or (2) slow dynamics. Weak or cooperative interactions typically require at least one species to be at micromolar or higher concentration, but far-field techniques cannot resolve fluorescent single molecules at concentrations higher than approximately 100 nM. Slow processes require long observation times, but surface immobilization often disrupts molecular structure or dynamics; freely diffusing molecules are typically observed for <1 ms.
The goals of imaging in the presence of background fluorescence and imaging in solution for long times are often seen as being in tension with each other. An approximate criterion for distinguishing a single fluorophore above background is that there should be no more than one fluorophore, on average, within a detection volume. Techniques with a smaller detection volume can detect single molecules in the presence of a higher background concentration. Among common imaging techniques, the maximum concentrations for detecting single molecules are ranked: zero-mode waveguides2
> total internal reflection fluorescence (TIRF)3
> confocal > epifluorescence. (See .) However, a smaller detection volume leads to a shorter diffusion-limited transit time. The observation times for freely diffusing molecules in these same imaging techniques are ranked: epifluorescence > confocal > TIRF > zero-mode waveguides.
Comparison of Single-Molecule Imaging Techniquesa
A strategy to circumvent this trade-off is to immobilize single molecules in confined volumes without direct surface attachment. This idea has been applied to study single molecules in liposomes,4,5
and the anti-Brownian electrokinetic trap (ABEL trap),8,9
but these systems are complex to build and operate.
We developed a simple device that dramatically improves imaging of single biomolecules. The device improves background rejection when imaging surface-immobilized molecules, and it increases observation time when imaging freely diffusing molecules. The convex lens-induced confinement (CLIC) system restricts molecules to a fluid film with nanometer dimensions perpendicular to the imaging plane. The confinement reduces the vertical dimension of the imaging volume and, thereby, improves background rejection. The confinement also keeps freely diffusing single molecules in the focal plane of the microscope and, thereby, increases imaging time. Additionally, one can use the CLIC system to determine the size of macromolecules because macromolecules are excluded from regions where the molecular diameter is larger than the depth of confinement.
In its simplest incarnation, the CLIC device consists of a plano-convex lens, curved side down, resting on top of a coverslip (). The region surrounding the point of contact is imaged in an inverted fluorescence microscope. The lens-coverslip gap height varies smoothly from zero at the point of contact, to hundreds of micrometers at positions far from the point of contact, according to
is the distance from the point of contact and R
is the radius of curvature of the lens (). This simple formula agrees quantitatively with in situ calibration (see Supporting Information Method 6
). For example, in a 100 μm × 100 μm field of view centered on the point of contact, with a 100 mm focal length lens (R
= 46 mm), the gap varies from 0 to 27 nm. Optical measurements at a series of points spaced by micrometers lead to information on molecular properties at a series of confinements spaced by nanometers.
Schematic of the CLIC device. The side view and front view show (a) counterweight, (b) micrometer, (c) rod, (d) jewel bearing, (e) XYZ translation stage, (f) optical flat and lens, (g) PDMS gasket, and (h) coverslip.
Figure 2 CLIC measurements of immobilized fluorescent objects in the presence of freely diffusing fluorophores. (a) Schematic of sample geometry. (b) Signal-to-background ratio as a function of displacement from the point of contact for surface-immobilized fluorescent (more ...)
We constructed an apparatus in which a micrometer and jewel bearing are used to raise and lower a lens from a coverslip surface in a controlled manner. We present single-molecule measurements that demonstrate the capabilities of the CLIC device. These include: (a) measurements on single immobilized molecules in the presence of a high concentration of freely diffusing fluorescent molecules; (b) counting of transmembrane proteins in freely diffusing lipid vesicles; and (c) direct mechanical measurements of size and compressibility of double stranded DNA.