Abstract
Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.
Highlights
All modalities of fluorescence microscopy face a practical limit to the number of photons they can collect from a given sample, and must make tradeoffs within this “photon budget” among spatial and temporal resolution, photodamage, and depth of imaging
To demonstrate the utility of spatiotemporal rank filtering for 3D imaging of living biological tissue, we collected a z-stack extending 200 μm down from the cortex of a mouse lymph node containing T cells labeled with Violet Proliferation Dye 450 (VPD-450), a typical chemical dye used in imaging studies
We initially collected 30 frames at each slice, saved individually, so that we could compare on the same data 5-frame averaging (5-FA), 5-frame averaging followed by spatial rank filtering (5-FA-SR), 5-frame spatiotemporal rank filtering (5-FSTR), which performs the rank on an n by n array of adjacent pixels taken from all 5 frames, and a higher quality frame averaging image with 6x as many frames (i.e. 30) representing an outermost practical limit of frame-averaging (30-FA) (Fig 1A)
Summary
All modalities of fluorescence microscopy face a practical limit to the number of photons they can collect from a given sample, and must make tradeoffs within this “photon budget” among spatial and temporal resolution, photodamage, and depth of imaging. Two-photon laser scanning microscopy (2P-LSM) [1] has enabled imaging of the behavior of mammalian cells in vivo [2] and is the method of choice for live imaging deep into scattering tissues including lymph nodes, lung, pancreas, and brain. The photon budget in 2P-LSM in vivo is an especially important consideration since photodamage, which compromises viability and can alter cell behavior, responds to increases in excitation power at higher order (i.e. supra-quadratic) rates compared to fluorescence emission [3,4]. PLOS ONE | DOI:10.1371/journal.pone.0150430 March 3, 2016
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
