Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy

Abstract

Fluorescence imaging is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the tradeoff between imaging speed and sensitivity 1 is problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues 2 , and cells flowing at high speed 3 . We report a solution that achieves real-time pixel readout rates one order of magnitude faster than a modern electron multiplier charge coupled device (EMCCD) -the gold standard in high-speed fluorescence imaging technology 4 . Deemed fluorescence imaging using radiofrequency-multiplexed excitation (FIRE), this approach maps the image into the radiofrequency spectrum using the beating of digitally synthesized optical fields. We demonstrate diffraction-limited confocal fluorescence imaging of stationary cells at a frame rate of 4.4 kHz, as well as fluorescence microscopy in flow at a throughput of approximately 50,000 cells per second.

Physical principles for scalable neural recording

“…With a known 3D pattern of excitation light, wavefront coding can be applied to 3D fluorescence microscopy without scanning using a 2D detector array (Quirin et al, 2013). Emerging, alternative strategies rely on tagging emissions from different sources with distinguishable modulation patterns (Yin, 2006; Wu et al, 2006; Wang et al, 2012; Diebold et al, 2013; Ducros et al, 2013), or precisely controlling and tracking the timing of light emissions (Cheng et al, 2011). Optical techniques thus achieve signal separation by multiplexing spatially (e.g., direct imaging) or temporally (e.g., beam scanning), or often by a combination of the two.…”

“…Light emissions from distinct locations can be tagged with distinguishable time-domain modulation patterns, and the emission time-series for each source can later be decoded from the summed signal resulting from scattering (Wu et al, 2006; Yin, 2006; Cheng et al, 2011; Wang et al, 2012; Diebold et al, 2013; Ducros et al, 2013). For example, ultrasound encoding (Wang et al, 2012; Judkewitz et al, 2013), which frequency-tags light emissions from a known location via a mechanical Doppler shift of the emitter (Mahan et al, 1998), provides a generic mechanism to sidestep problems of elastic optical scattering, although it requires distinguishing MHz frequency modulations in THz light waves (part per million frequency discrimination).…”

Three-dimensional single-pixel imaging of incoherent light with spatiotemporally modulated illumination

“…While there are several other microscopy techniques that have similar modulation patterns [6,11,12,16], we will focus our discussion on the illumination patterns utilized for CHIRPT. We note, however, that other forms of modulation can be readily extended from the analysis of CHIRPT [13].…”

Radiofrequency encoded angular-resolved light scattering

“…9 how SEALS encodes the angular scattering profile into the spectrum of a broadband optical pulse, this technique, termed Radio-frequency Encoded Angular-resolved Light Scattering (REALS), uses a one-to-one radiofrequency-toangle mapping of a continuous wave (CW) laser to encode the scattering profile into the radio-frequency (RF) domain. Upon detection by a single-pixel photomultiplier tube (PMT), the scattering profile can be recovered using an electrical spectrum analyzer (ESA) or the signal can be digitized and analyzed in real-time using digital frequency domain techniques such as the fast Fourier transform (FFT).…”

“…The beam in the first arm is diffracted into multiple beams (RF beams) using a wide bandwidth AO deflector (AOD), driven by a radiofrequency comb produced by direct digital synthesis (DDS). 9 The time-bandwidth product (TBP) of the AOD dominates the angular resolution of the system. Through the acousto-optic interaction, diffracted optical beams are shifted in both output angle and optical frequency by the corresponding comb line frequencies.…”