Lists

Wednesday, August 07, 2024

Pixel-level programmable regions-of-interest for high-speed microscopy

Zhang et al. from MIT recently published a paper titled "Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy" in Nature Communications.

Abstract: High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2–3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

 

a Pixels within an ROI capture spatiotemporally-correlated physiological activity, such as signals from somatic genetically encoded voltage indicators (GEVI). b Simulated CMOS pixel outputs with uniform exposure (TE) face the trade between SNR and temporal resolution. Short TE (1.25 ms) provides high temporal resolution but low SNR. Long TE (5 ms) enhances SNR but suffers from aliasing due to low sample rate, causing spikes (10 ms interspike interval) to be indiscernible. Pixel outputs are normalized row-wise. Gray brackets: the zoomed-in view of the pixel outputs. c Simulated pixel outputs of the PE-CMOS. Pixel-wise exposure allows pixels to sample at different speeds and phases. Two examples: in the staggered configuration, the pixels sample the spiking activity with prolonged TE (5 ms) at multiple phases with offsets of (Δ = 0, 1,25, 2.5, 3.75 ms). This configuration maintains SNR and prevents aliasing, as the interspike interval exceeding the temporal resolution of a single phase is captured by phase-shifted pixels. In the multiple exposure configuration, the ROI is sampled with pixels at different speeds, resolving high-frequency spiking activity and slow varying subthreshold potentials that are challenging to acquire simultaneously at a fixed sampling rate. d The PE-CMOS pixel schematic with 6 transistors (T1-T6), a photodiode (PD), and an output (OUT). RST, TX, and SEL are row control signals. EX is a column signal that controls pixel exposure. e The pixel layout. The design achieves programmable pixel-wise exposure while maximizing the PD fill factor for high optical sensitivity.

 

a Maximum intensity projection of the sCMOS (Hamamatsu Orca Flash 4.0 v3) and the PE-CMOS videos of a cultured neuron expressing the ASAP3 GEVI protein. b ROI time series from the sCMOS sampled at 800 Hz with pixel exposure (TE) of 1.25 ms. Black trace: ROI time series. Gray trace: the time series each with 1/4 pixels of the ROI. Plotted signals are inverted from raw samples for visualization. c simultaneously imaged ROI time series of the PE-CMOS. Colored trace: the time series of phase-shifted pixels at offsets (Δ) of 0, 1.25, 2.5, and 3.75 ms each contain 1/4 pixels of the ROI. All pixels are sampled at 200 Hz with TE = 5 ms. Black trace: the interpolated ROI time series with 800 Hz equivalent sample rate. Black arrows: An example showing a spike exceeding the temporal resolution of a single phase is captured by phase-shifted pixels. Black circles: an example subthreshold event barely discernable in sCMOS is visible in the pCMOS output. d, e, f: same at panels (a, b, c) with an example showing a spike captured by the PE-CMOS but not resolvable in the sCMOS output due to low SNR (marked by the magenta arrow). g, h comparison of signal quality from smaller ROIs covering parts of the cell membrane. Gray boxes: zoomed-in view of a few examples of putative spiking events. i SNR of putative spikes events from ROIs in panel (g). A putative spiking event is recorded when the signals from either output exceed SNR > 5. Data are presented as mean values +/- SD, two-sided Wilcoxon rank-sum test for equal medians, n = 93 events, p = 2.99 × 10-24. The gain is calculated as the spike SNR in the PE-CMOS divide by the SNR in the sCMOS. All vertical scales of SNR are 5 in all subfigures.

a The intracellular potential of the cell and the ROI GEVI time-series of the PE-CMOS and sCMOS. GEVI pulse amplitude is the change in GEVI signal corresponding to each current injection pulse. It is measured as the difference between the average GEVI intensity during each current pulse and the average GEVI intensity 100 ms before and after the current injection pulse. GEVI pulse amplitude is converted into SNR by dividing the noise standard deviation. b max. projection of the cell in PE-CMOS and sCMOS. c zoomed in view of the intracellular voltage and GEVI pulses in (a). The red arrow indicates spike locations identified from the intracellular voltage. The black arrows indicate a time where intracellular potential shows a flat response when the GEVI signals in both PE-CMOS and sCMOS exhibit significant amplitude variations. These can be mistaken for spiking events. d zoomed in view of (c) showing the PE-CMOS trace can resolve two spikes with small inter-spike interval, while sCMOS at 800 Hz and 200 Hz both fail to do so. The blue arrows point to the first spike invoked by the current pulse. While the sharp rising edges make them especially challenging for image sensors to sample, the PE-CMOS can preserve their amplitudes better the sCMOS.
 

a Maximum intensity projection of the PE-CMOS videos, raw and filtered (2 × 2 spatial box filter) output at full spatial resolution. Intensity is measured by digital bits (range: 0–1023). b Maximum intensity projection divided into four sub-frames according to pixel sampling speed, each with 1/4 spatial resolution. c The ROI time series from pixels of different speeds (colored trace). Black trace: a 1040 Hz equivalent signal interpolated across all ROI pixels. d Fast sampling pixels (520 Hz) resolves high-SNR spike bursts. e–f Pixels with more prolonged exposure (TE = 2.8–5.7 ms) improves SNR to detect weak subthreshold activity (black arrow) and (f) low SNR spike. The vertical scale of SNR is 10 unless otherwise noted.


Open access article link: https://www.nature.com/articles/s41467-024-48765-5

No comments:

Post a Comment

All comments are moderated to avoid spam and personal attacks.