BioPhotonics Preview for July/August 2024

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Here is your first look at the editorial content for the upcoming July/August issue of BioPhotonics.

STED Microscopy

First developed in 1994 by Stefan Hell and his colleagues, stimulated emission depletion (STED) microscopy excites a small region with a focused laser beam, then uses a second laser beam to switch off surrounding fluorophores, creating a "donut hole" of high resolution. One advantage of STED has historically been its relatively simple instrumentation (pulsed laser, avalanche photodiode, and fluorescent dyes) but refining its application has been challenging. Leica has developed the TauSTED approach, where the fluorescence lifetime information related to the point spread function of specific molecules can be extracted. Hell's group has worked on a technique called MINSTED, wherein the exact position of a fluorophore can be tracked with a finite number of photons, which can locate fluorophores attached to specific DNA strands. At the University of Central Florida, they have shown that while photobleaching has historically been a significant issue for STED, it is possible to denoise the images, allowing for illumination to be applied for only microseconds. This could open the door to the development of a vast library of STED images for the life science community.

Key Technologies: STED microscopy

Raman Spectroscopy and Blood Analysis

Recent technological advancements have enabled researchers to analyze blood for a variety of biomarkers simultaneously, significantly enhancing diagnostic capabilities. Raman spectroscopy-based blood diagnostics represent an extremely innovative field of study. This technique allows for a comprehensive examination of cellular components and plasma with just a few microliters of sample. Efforts are now underway to adapt these concepts for point-of-care diagnostics. This includes customized sample preparation, intelligent measurement workflows, functional bioassays, and high-throughput devices. The point-of-care approach is further enhanced by machine learning pipelines that automatically analyze spectroscopic data and present the findings as straightforward medical diagnostics. This article will primarily focus on Raman spectroscopic methods for point-of-care blood diagnostics.

Key Technologies: Raman spectroscopy

Microscope Objective Design

Microscope objectives are critical for a vast range of biomedical imaging applications ranging from multiphoton microscopy to differential interference contrast microscopy. The high level of accuracy required for these systems leads to unique challenges in the design of microscope objectives appropriate for life science and biomedical research as opposed to other optical assemblies like factory automation lenses. Active alignment and other advanced assembly techniques are required to achieve the tight tolerances needed, and a focus on chromatic aberration correction results in high performance across the desired wavelength range. Understanding the fundamentals of objective design will help you better select the right objective for your application and understand how to optimally balance objective specifications with the rest of the system requirements.

Key Technologies: Microscopy, microscope objectives

Fluorescence Lifetime Imaging

FLIM is a powerful technique that provides a totally new dimension to quantitate the fluorescent probe, in addition to its steady-state intensity and spectral profiles. With peculiar selectivity of probes, FLIM can provide quantitative information of the probe microenvironment such as ions, pH, oxygen content, electrical signals, and index of refractions. FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions in live specimens. Turn-key commercial FLIM instruments are mature, making the technique easily accessible; however, a major challenge is still how to analyze and interpret FLIM data. the phasor plots approach has been demonstrated to be an effective solution. Here, we describe several quantitative tools using the phasor plots in a software multi-image phasor analysis (MiPA) module; we demonstrate their utilities with examples for various applications including time-resolved multiplexing, label-free NADH imaging, STED superresolution imaging, and FRET.

Key Technologies: Fluorescence lifetime imaging, Forster Resonance Energy Transfer, time-resolved multiplexing, superresolution imaging

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Published: May 2024

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