Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

Abstract

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed ''optical switches.'' This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.high-contrast ͉ optical switches ͉ "ac"-imaging ͉ fluorescence microscopy U nderstanding the molecular basis for the regulation of complex biological processes such as cell motility and proliferation requires analysis of the distribution and dynamics of protein interactions within living cells in culture and in intact tissue (1). Tremendous advances have been made toward the development of new optical probes (2, 3) and imaging techniques that are capable of detecting proteins down to the level of single molecules (4-11). However, in living cells, such detection is compromised by autofluorescence, which can amount to several thousand equivalents of fluorescein per cell (12), as well as by light scattering (13). A major challenge in live-cell imaging, therefore, is to develop classes of probes and imaging techniques that are capable of resolving fluorescence signals from synthetic probes or genetically encoded fluorescent proteins in living cells and tissue against large background signals that may vary in time and space.A simple and highly-effective approach for isolating a specific fluorescence signal from a large background is to reversibly modulate the fluorescence intensity of only a probe of interest that is bound to a specific protein by applying a uniform, rapid and specific perturbation (e.g., a change in temperature (14), pressure (15), or voltage (16) to which that probe is uniquely attuned. The modulated fluorescence can be isolated from other steady sources of background fluorescence by lock-in detection, making it possible to specifically extract the probe fluoresc...

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

“…1f,g). In order to differentiate neighboring nanoareas in the presence of background noise, a sufficiently large modulation signal can be achieved either by averaging the modulation over several periods (similarly to lock-in detection, which is also based on the periodic recurrence of signals in the presence of a large background 14,15 ) or by using ExPAN.…”

“…In addition, the periodic recurrence of the signals allows the signal-to-noise ratio to be improved by averaging several periods and thereby enhancing the ability to distinguish nanoareas in a manner similar to lock-in detection 14,15 . Unlike techniques based on single-molecule detection, in which it is essential to detect only very isolated photoswitchable single molecules, SPoD also allows bundled signals of several standard fluorescent molecules with different modulation phases to be distinguished in adjacent nanoareas.…”

Hybridization kinetics is different inside cells

“…As a side note, the herein described optical lock-in (21) has to be distinguished from a more recent method with a similar name (44,45) (21). For temperature measurements, fluorescence amplitudes were translated to temperature changes via the Cy5 calibration.…”

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

“…Stochastic techniques using photoswitchable probes have also been developed such as photo-activated localization microscopy (fPALM) (7), stochastic optical reconstruction microscopy (STORM) (8), PALM (9), and variants thereof (10,11). The development of switchable fluorescent probes also triggered the emergence of new contrast enhancing techniques such as optical lock-in detection (OLID) (12). Even though OLID provides fast imaging with enhanced contrast, it lacks super-resolution capability.…”