Volume 29 Issue 10 | Journal of Biomedical Optics

Journal of Biomedical Optics

VOL. 29 · NO. 10 | October 2024

ISSUES IN PROGRESS

IN PROGRESS

SPIE publishes accepted journal articles as soon as they are approved for publication. Journal issues are considered In Progress until all articles for an issue have been published. Articles published ahead of the completed issue are fully citable.

IN THIS ISSUE

Imaging (1)

Microscopy (1)

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Imaging

Dynamic multispectral NIR/SWIR for in vivo lymphovascular architectural and functional quantification

Christopher Hansen, Jaidip Jagtap, Abdul Parchur, Gayatri Sharma, Shayan Shafiee, Sayantan Sinha, Heather Himburg, Amit Joshi

Journal of Biomedical Optics, Vol. 29, Issue 10, 106001, (September 2024) https://doi.org/10.1117/1.JBO.29.10.106001

TOPICS: Lymphatic system, Short wave infrared radiation, Biological imaging, In vivo imaging, Fluorescence imaging, Imaging systems, Image resolution, Quantum imaging, Silver, Spatial resolution

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Microscopy

Simultaneous assessment of NAD(P)H and flavins with multispectral fluorescence lifetime imaging microscopy at a single excitation wavelength of 750 nm

Boris Yakimov, Anastasia Komarova, Elena Nikonova, Artem Mozherov, Liubov Shimolina, Marina Shirmanova, Wolfgang Becker, Evgeny Shirshin, Vladislav Shcheslavskiy

Journal of Biomedical Optics, Vol. 29, Issue 10, 106501, (September 2024) https://doi.org/10.1117/1.JBO.29.10.106501

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Significance

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications.

Aim

We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells.

Approach

We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids.

Results

The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins.

Conclusions

The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

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