Hyperdimensional Imaging Contrast Using an Optical Fiber

Abstract

Fluorescence properties of a molecule can be used to study the structural and functional nature of biological processes. Physical properties, including fluorescence lifetime, emission spectrum, emission polarization, and others, help researchers probe a molecule, produce desired effects, and infer causes and consequences. Correlative imaging techniques such as hyperdimensional imaging microscopy (HDIM) combine the physical properties and biochemical states of a fluorophore. Here we present a fiber-based imaging system that can generate hyper-dimensional contrast by combining multiple fluorescence properties into a single fluorescence lifetime decay curve. Fluorescence lifetime imaging microscopy (FLIM) with controlled excitation polarization and temporally dispersed emission can generate a spectrally coded, polarization-filtered lifetime distribution for a pixel. This HDIM scheme generates a better contrast between different molecules than that from individual techniques. This setup uses only a single detector and is simpler to implement, modular, cost-efficient, and adaptable to any existing FLIM microscope. We present higher contrast data from Arabidopsis thaliana epidermal cells based on intrinsic anthocyanin emission properties under multiphoton excitation. This work lays the foundation for an alternative hyperdimensional imaging system and demonstrates that contrast-based imaging is useful to study cellular heterogeneity in biological samples.

Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution

“…This makes it compatible with any wide-field technique including light-sheet, − TIRF illumination, − and spinning-disc confocal microscopy. It also makes EO-FLIM naturally suited to applications where certain detector characteristics are desired or where multiple detection dimensions are combined. ,,,, Applications in high-throughput single-molecule imaging and light-sheet microscopy − appear particularly promising. Further, we anticipate that next-generation camera sensors will allow impressive capabilities when combined with nanosecond EO modulation.…”

Resonant Electro-optic Imaging for Microscopy at Nanosecond Resolution

“…This makes it naturally suited to applications where certain detector characteristics are desired or where multiple detection dimensions are used. 5,7,45,46 Applications in high-throughput single-molecule imaging and light-sheet microscopy appear particularly promising. Further, we anticipate that next-generation camera sensors will enable impressive capabilities combined with nanosecond modulation.…”

CASPI: collaborative photon processing for active single-photon imaging

“…These microscopes use pulsed femtosecond lasers operating at a repetition rate 8 × 10 7 and 720 nm dichroic cut-off filter for separating fluorescence. The microscopes are designed for fluorescence lifetime imaging, and the FLIM data were collected using time-correlated single-photon counting (TCSPC) electronics 67 , 68 . The photons were collected using a photosensitive GaAsP PMT (H7422, Hamamatsu), and single-photon timings were determined by the SPC-150 timing module (Becker-Hickl GmbH, Berlin).…”