Real-time dynamic range and signal to noise enhancement in beam-scanning microscopy by integration of sensor characteristics, data acquisition hardware, and statistical methods

Abstract

Despite the ubiquitous use of multi-photon and confocal microscopy measurements in biology, the core techniques typically suffer from fundamental compromises between signal to noise (S/N) and linear dynamic range (LDR). In this study, direct synchronous digitization of voltage transients coupled with statistical analysis is shown to allow S/N approaching the theoretical maximum throughout an LDR spanning more than 8 decades, limited only by the dark counts of the detector on the low end and by the intrinsic nonlinearities of the photomultiplier tube (PMT) detector on the high end. Synchronous digitization of each voltage transient represents a fundamental departure from established methods in confocal/multi-photon imaging, which are currently based on either photon counting or signal averaging. High information-density data acquisition (up to 3.2 GB/s of raw data) enables the smooth transition between the two modalities on a pixel-by-pixel basis and the ultimate writing of much smaller files (few kB/s). Modeling of the PMT response allows extraction of key sensor parameters from the histogram of voltage peak-heights. Applications in second harmonic generation (SHG) microscopy are described demonstrating S/N approaching the shot-noise limit of the detector over large dynamic ranges.

Neuronal imaging with ultrahigh dynamic range multiphoton microscopy

“…Compared to multi-exposure 15 , multi-signal-attenuation 16 , or post-detection 17 strategies for achieving high dynamic range, our technique provides the added benefits of limiting photobleaching or phototoxicity in the sample ( P max is set to no higher than the illumination power P 0 normally used in standard imaging – see Supplementary Information), and reducing potential of damage to the detector itself caused by sudden signal transients ( S is prevented from being larger than S 0 ). It should be noted that active illumination can also be applied to single photon scanning microscopy (e.g.…”

Synchronous-digitization for video rate polarization modulated beam scanning second harmonic generation microscopy

“…All experiments were performed on a custom built microscope described in detail previously 9,13–15 . In short, a 80 MHz ~100 fs MaiTai Ti:Sapphire laser (SpectraPhysics) operating at 800nm was used, with average powers between 20–220mW at the sample.…”

Exciton Coupling Model for the Emergence of Second Harmonic Generation from Assemblies of Centrosymmetric Molecules

“…SHG microscopy measurements of TAQ crystals were performed using an instrument described previously. − In brief, all images were acquired with a built-in-house beam scanning SHG microscope. Beam scanning was performed with a resonant vibrating mirror (∼8 kHz, EOPC) along the fast-axis scan, and a galvanometer (Cambridge) for slow-axis scanning.…”