fluorescence-lifetime-macro-imager-for-biomedical-applications

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

The University of Western Australia

The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout rate of 80 megapixels per second. In this study, we coupled the camera to an image intensifier, a 760 nm emission filter and a 50 mm lens, and with a super-bright 627nm LED providing pulsed excitation of a 18 × 18 mm sample area. The resulting macro-imager with compact and rigid optical alignment of its main components was characterised using planar phosphorescent O sensors and a resolution plate mask. Several acquisition and image processing algorithms were evaluated to optimise the system resolution and performance for the wide-field PLIM, followed by imaging a variety of phosphorescent samples. The new PLIM system looks promising, particularly for phosphorescence lifetime-based imaging of O in various chemical and biological samples.

New luminescence lifetime macro-imager based on a Tpx3Cam optical camera.

O PLIM microscopy was employed in various studies, however current platforms have limitations in sensitivity, image acquisition speed, accuracy and general usability. We describe a new PLIM imager based on the Timepix3 camera (Tpx3cam) and its application for imaging of O concentration in various tissue samples stained with a nanoparticle based probe, NanO2-IR. Upon passive staining of mouse brain, lung or intestinal tissue surface with minute quantities of NanO2-IR or by microinjecting the probe into the lumen of small or large intestine fragments, robust phosphorescence intensity and lifetime signals were produced, which allow mapping of O in the tissue within 20 s. Inhibition of tissue respiration or limitation of O diffusion to tissue produced the anticipated increases or decreases in O levels, respectively. The difference in O concentration between the colonic lumen and air-exposed serosal surface was around 140 µM. Furthermore, subcutaneous injection of 5 µg of the probe in intact organs (a paw or tail of sacrificed mice) enabled efficient O imaging at tissue depths of up to 0.5 mm. Overall, the PLIM imager holds promise for metabolic imaging studies with various ex vivo models of animal tissue, and also for use in live animals.

Mapping O concentration in ex-vivo tissue samples on a fast PLIM macro-imager.

With the invention of the Atomic Force Microscope (AFM) in 1986 and the subsequent developments in liquid imaging and cellular imaging it became possible to study the topography of cellular specimens under nearly physiological conditions with nanometric resolution. The application of AFM to biological research was further expanded with the technological advances in imaging modes where topographical data can be combined with nanomechanical measurements, offering the possibility to retrieve the biophysical properties of tissues, cells, fibrous components and biomolecules. Meanwhile, the quest for breaking the Abbe diffraction limit restricting microscopic resolution led to the development of super-resolution fluorescence microscopy techniques that brought the resolution of the light microscope comparable to the resolution obtained by AFM. The instrumental combination of AFM and optical microscopy techniques has evolved over the last decades from integration of AFM with bright-field and phase-contrast imaging techniques at first to correlative AFM and wide-field fluorescence systems and then further to the combination of AFM and fluorescence based super-resolution microscopy modalities. Motivated by the many developments made over the last decade, we provide here a review on AFM combined with super-resolution fluorescence microscopy techniques and how they can be applied for expanding our understanding of biological processes.

Article	Year
New luminescence lifetime macro-imager based on a Tpx3Cam optical camera. Biomedical optics express\| PubMed ID: 32010501	2020
Mapping O concentration in ex-vivo tissue samples on a fast PLIM macro-imager. Scientific reports\| PubMed ID: 33149165	2020
How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology? Nanoscale\| PubMed ID: 33346312	2021

Liisa M. Hirvonen

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