Fluorescence lifetime imaging (FLIM) is a powerful technique often employed in medical diagnostics research. It uses precision lasers, fluorescent dyes, and advanced recording devices. FLIM allows medical professionals and researchers to monitor living cells, sub-cell interactions, and the proteins responsible for them with greater accuracy and precision. This makes FLIM a valuable tool for clinical diagnosis and offers a new, molecular-level understanding of immune responses, allergies, and even cancer.
Fluorescence lifetime imaging is empowering medical professionals with a sub-cell view of problems even as they emerge. Image Credt: Avantier Inc.
But what is going on when an image using FLIM is generated? The bulk of the key information your image is based on refers to the differences in the fluorescence lifetime of excited fluorophore dyes, which may be used to tag various proteins or parts of proteins.
To put it simply, fluorescence lifetime microscopy can be thought of as scanning a sample under a microscope with a laser beam. Fluorescence can be measured at every position across the entire sample. Various parameters of the biochemical sample, such as the pH, ion concentration, and fluorophore concentration, determine the rate at which the fluorescence intensity decreases; the refractive index of the environment also plays its part.
What is time-correlated single-photon counting?
Most fluorescence lifetime imaging today is conducted using time-correlated single-photon counting (TCSPC). TCSPC enables the quantification of the time that passes between an excitation pulse (by laser) and one emitted photon. The photon’s arrival is measured by a sensitive detector which generates a separate electric pulse at each arrival interval. This detector may be a single photon avalanche photodiode (SPAD) or a photo-multiplier tube (PMT).
When a considerable number of times have been sufficiently recorded, the photon counts can be categorized by arrival time into an intensity-based histogram. Current methods facilitate the recording of millions of start-stop times over a reasonably sized interval.
Time-correlated single-pulse counting can be used to measure lifetimes as short as a few pico seconds in fluorescence-lifetime imaging. Image Credt: Avantier Inc.
What is the gating method?
The gating method is an alternative way of conducting FLIM. In this method, part of the laser pulse traveling toward the sample is reflected onto a photodiode via a dichroic mirror. The photodiode triggers a delay generator that regulates a gated optical intensifier (GOI) positioned in front of a CCD (charge-coupled device) detector.
Each time the GOI opens, it enables detection for a brief time interval. When fluorescence emission is acquired after numerous delay times, this system can record a detailed view of the sample's fluorescence decay time range. Today’s integrated intensified CCD cameras facilitate sub-nano-second resolution FLIM.
Fluorescence resonance energy transfer (FRET) microscopy
FLIM alone cannot achieve zoomed-in viewing of protein-protein interactions; this is where Fluorescence Resonance Energy Transfer (FRET) Microscopy comes in handy.
When two fluorophores are no more than 10 nanometers apart, excitation can be transmitted between them without photons. This transfer is distance-dependent, with an increase in the efficiency occurring at a rate proportional to the inverse sixth power of the separation between the molecules. FRET is often used in combination with FLIM to establish the correct spatial proximity of proteins and molecules.
Biomedical imaging and diagnostics
Since the birth of modern medicine, traditional imaging techniques have been a central component to making accurate medical diagnoses, yet, they are limited in the depth of information they can provide about biological processes and the onset of diseases.
The development of improved imaging techniques that allow medical practitioners and researchers to observe biological processes in situ will contribute to a deeper understanding of pathological and normal tissue composition, morphology, and function.
Imaging techniques for biomedical applications is a fast-emerging field, and the advances made in recent years have been great. Approaches in fluorescent lifetime imaging, for instance, have made significant progress, which has transformed several areas of medical diagnostics. A combination of high-power lasers, versatile beam scanners, sensitive detectors, and high-resolution cameras has allowed researchers to view biological tissue in vivo and gain invaluable insights into disease and healthy physiology.
Optical imaging techniques: Viewing tissue at depth
Due to its dense, inhomogeneous nature, biological tissue is often opaque and inherently difficult to image. Tissue scattering has made deep tissue imaging extremely difficult in the past. However, two-photon fluorescence microscopy has been successful at generating high-resolution imaging even at deeper tissue levels, and today, recent advances are facilitating high-quality imaging capture at a level deeper than ever before.
Avantier Inc. is at the vanguard of imaging systems and optical solutions and has an excellent track record of providing medical practitioners, researchers, and industry clients with the optics needed to push the envelope of high-performance imaging. Contact Avantier today for more information.
About Avantier Inc.
Avantier Inc. is an unparalleled leader in providing imaging systems solutions and optical solutions.
They offer advanced precision custom optical design, optical engineering, optical lens assembly, rapid optical proto-typing, image processing and manufacturing services.
Avantier is ISO 9001:2015, ISO 13485:2016, and ISO 14001:2015 certified.
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