Hyperspectral multiphoton microscopy for in vivo visualization of multiple, spectrally overlapped fluorescent labels

Abstract

The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

“…Notably, this is a critical yet straightforward add-on obviating the need for complicated spectral unmixing or post hoc image processing. In contrast, multispectral imaging techniques employ distinct fluorescent biosensors (genetically encoded or chemical-based) [ 22 , 23 ], usually in the same cells. Although very informative, technical challenges (i.e., combining suitable filter settings and establishing complicated spectral unmixing algorithms) undermine implementing these methods.…”

“…Thus, one concern about this approach that is still elusive whether a 1:1:1 ratio remains constant over a longer time of passaging, freezing, and thawing cycles. Co-culturing cells with fluorescent reporters is a favored method employed in many studies [ 22 , 23 , 25 ]; however, to our knowledge, such an approach as presented in this study has not been shown elsewhere as a multiparametric imaging technique.…”

Phytoglycogen Encapsulation of Lanthanide‐Based Nanoparticles as an Optical Imaging Platform with Therapeutic Potential

“…[ 53 ] This technique is particularly attractive in combination with optical multiplexing as it can significantly increase the throughput of (bio)imaging by tracking multiple targets, such as fluorescent proteins or luminescent nanoparticles. [ 54,55 ] The bright field optical microscopy image (Figure 2A2) shows a region containing UCNP‐PGLY beads of similar morphologies. However, when hyperspectral mapping was performed at the regions of interest indicated in green, blue and cyan (magnified in Figure 2B/C1–4 for better visualization), the characteristic spectral profiles of UCNP‐2, UCNP‐3, and UCNP‐4 were unequivocally identified (Figure 2D1–4).…”

In vivo simultaneous nonlinear absorption Raman and fluorescence (SNARF) imaging of mouse brain cortical structures

“…First, TPEF spectra are typically broad (~50 nm), which can make detection of more than 3 fluorophores in vivo difficult. Spectral unmixing has recently been developed to address this challenge, but it still comes at the expense of system complexity and cost due to the use of multiple lasers and computational unmixing 43 . Second, transgenic animals with multiple fluorescent proteins encoding different cell types require extensive breeding, which brings substantial costs in time and resources.…”