BioPhotonics Preview - January/February 2023

Light Sources and Imaging

LEDs and Top Hat Illumination Benefits Microscopy, Patterned Projection, PCR, and More Many applications desire even illumination across some plane of interest, such as excitation of fluorophores across specimen areas in fluorescence microscopy, patterned illumination in super-resolution microscopy, illumination in quantitative PCR instruments, and projection in maskless lithography and cinematography. Classical methods of illumination (such as Köhler and critical) tend to fall short in one or some other aspect of illumination. For example, while Köhler illumination homogenizes hot spots arising from nonuniform light sources, it tends to produce a globally non-uniform “dome-shaped” illumination profile. On the other hand, critical illumination can produce globally even illumination but tends to have localized hot/cold spots depending on the nature of the light source. In contrast to these classical methods, a “ modern classical” approach uses LEDs combined with aberration theory, Fourier optics, and non-imaging optics, producing flat “top hat” illumination distributions across any plane of interest – even if the illumination is oblique. Here, we review classical methods of illumination and highlight a new technology that overcomes their shortcomings.

Key Technologies: LEDs, microscopy, Kohler illumination, critical illumination, collimated illumination, light pipe illumination, fly's eye illumination, top hat illumination

Laser Scanning Microscopy

Advanced photonic components based on acousto-optics and other technologies address the speed and resolution limitations of confocal imaging, particularly for deeper in vivo imaging in neuroscience and intravital applications. Confocal laser scanning microscopy is a mainstay imaging method that provides high-resolution 3D images. For example, in neuroscience, it is commonly used to study both anatomy and activity of neural networks in the live mammalian (e.g., murine) brain in real time at single neuron spatial resolution. In many of these studies, scientists want to look deeper into the brain and monitor ever larger populations of neurons. Unfortunately, the information contained in these studies is often compromised by the speed and resolution limitations of confocal microscopy. Specifically, the speed is often limited by the speed of scanning the laser beam waist in three axes (xyz), relative to the sample. The resolution in deeper images frequently is limited by optical aberrations (wavefront distortions) due to the inhomogeneity of the tissue. In this article we will discuss how several advances in photonic devices and the way they are deployed can successfully address these limitations.

Key Technologies: confocal microscopy, acousto-optics, laser scanning microscopy, Michelson interferometer

Single-Molecule Spectroscopy

Infrared spectroscopy is a robust analytical technique that provides molecular information based on the interaction of infrared radiation with matter. Due to its unique fingerprinting capability, infrared spectroscopy has been widely used to interrogate the overall biochemistry of biological systems, which has led to the flourishing of the technique as a powerful physicochemical method with broad applications within the life sciences. Advances in instrumentation and the need for data acquisition speed and spatial resolution have contributed to the development of different infrared-based technologies over the years. More recently, a novel far-field pump-and-probe scheme, so-called optical photothermal infrared (O-PTIR) spectroscopy, has been proposed to acquire infrared chemical images with sub-micron spatial resolution in a fast manner. Here, we discuss the impacts, limitations, and future perspectives of such technology in the biomedical research field, including its applications as a single-cell microbial metabolomics tool as well as to the study of tissue diseases.

Key Technologies: infrared spectroscopy, microbial metabolomics, FTIR spectrometer, Raman spectroscopy

Optogenetics

Optogenetics is a novel neuroscience tool that facilitates studies of brain function by selectively activating photo-sensitive neurons and measuring induced brain activity. However, brain tissue heavily affects light illumination since scattering and absorption events occur deep into the brain. Computer Generated Holograms are employed for neural photostimulation as they provide arbitrary illumination patterns, but they still suffer from these events. Our approach resolves this limitation by engaging advanced optimization algorithms in the hologram design procedure. In particular, brain tissue is simulated as a series of parallel phase masks. Each phase mask corresponds to a different depth into the tissue and implies a shift onto the propagated light field based on the tissue properties. Our advanced algorithms consider the scattering effects of brain tissue in the hologram optimization process. As such, the specificity of holographic optogenetic stimulation is enhanced, fostering arbitrary neural illumination.

Key Technologies: Optogenetics, holography, AI, spatial light modulator, lithography

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