A confocal line-scanning FLIM microscope for fast biological imaging

Session

Poster Session

Authors

Anneliese Jarman (1), Ani A. Jose (1), Hanning Mai (2), Ahmet Erdogan (2), Conor Treacy (1), Neil Finlayson (2), Robert Henderson (2), Simon Poland (1)

Affiliations

1. King's College London
2. University of Edinburgh

Keywords

fluorescence, lifetime, microscopy, confocal, line-scanning, SPAD, TCSPC, 3D, dynamic

Abstract text

Fluorescence lifetime imaging (FLIM) microscopy is used to report on many characteristics within the cellular environment by measuring local variations in biological cells and tissues via extrinsic fluorescent probes and intrinsic fluorescent metabolic markers. Until recently the use of FLIM has been limited in the measurement of dynamic biological processes due to the need for high photon counts which are required to perform lifetime fitting. Traditionally TCSPC based approaches suffer from prolonged electronic dead time between successive photon arrival events, extending the exposure time required to collect adequate photons for decay fitting.  This can lead to pulse pile-up which results in short-skewed lifetime assignations. Although the integration of enhanced time-to-digital converter (TDC) electronics has significantly decreased the interval between successive pulses, the use of single beam scanning FLIM techniques still necessitates high-powered laser excitation with short pixel dwell times for rapid FLIM data collection, which can potentially perturb or damage the sample.

We report on the development of a simple confocal, line-scanning fluorescence lifetime microscope exploiting a novel SPAD line sensor with on-chip histogramming which will enable the optical interrogation of fast dynamic processes thanks to acquisition of up to 16.5 Giga-photon counts/s.

The system uses a cylindrical lens to generate a laser line focus and a single-axis scanner to generate a sweeping line across the sample. Fluorescence emission is collected by the same objective, de-scanned, and reprojected onto the blue/green sensitive portion of the linear array sensor.[1]Each pixel in the 512 x 2 pixel sensor is comprised of 2 x 8 SPADs and has its own dedicated 16-bit time-to-digital converter (TDC). In histogramming mode, the individual photon arrival times for image reconstruction can be passed into, and stored in, their bin block on the chip until the histograms are read off the chip after an externally triggered event.[2] For samples with low levels of fluorescence, the mass-parallelization of both fluorescent excitation and detection provides a great advantage over single-beam scanning FLIM technologies.

Additionally, the individually addressable SPADs can be switched on or off to produce custom sensor patterns: Two key uses for this functionality are separating the two pixel SPAD columns so that the pixel number can be doubled, and decreasing the width of the detector by turning off the outer rows. In this latter case, leaving just the middle row of SPADs active in each of the 512 pixels gives us a sensor width of approximately 0.6 Airy units, making the system fully confocal. Three-dimensional, and live, biological datasets will be presented to evidence the advanced functionality of the sensor in a fluorescence lifetime imaging context.

References

[1] Mai, H., Jarman, A., Erdogan, A.T., Treacy, C., Finlayson, N., Henderson, R.K. and Poland, S.P., 2023. Development of a high-speed line-scanning fluorescence lifetime imaging microscope for biological imaging. Optics Letters, 48(8), pp.2042-2045.

[2] Erdogan, Ahmet T., et al. "A CMOS SPAD line sensor with per-pixel histogramming TDC for time-resolved multispectral imaging." IEEE Journal of Solid-State Circuits 54.6 (2019): 1705-1719onfoc