Few-Photon Spectral Confocal Microscopy for Cell Imaging Using Superconducting Transition Edge Sensor.

Kazuki Niwa,Kaori Hattori,Daiji Fukuda

doi:10.3389/fbioe.2021.789709

Abstract

A superconducting transition edge sensor (TES) is an energy-dispersive single-photon detector that distinguishes the wavelength of each incident photon from visible to near-infrared (NIR) without using spectral dispersive elements. Here, we introduce an application of the TES technique for confocal laser scanning microscopy (CLSM) as proof of our concept of ultra-sensitive and wide-band wavelength range color imaging for biological samples. As a reference sample for wide-band observation, a fixed fluorescence-labeled cell sample stained with three different color dyes was observed using our TES-based CLSM method. The three different dyes were simultaneously excited by irradiating 405 and 488 nm lasers, which were coupled using an optical fiber combiner. Even when irradiated at low powers of 80 and 120 nW with the 405 and 488 nm lasers respectively, emission signals were spectrally detected by the TES and categorized into four wavelength bands: up to 500 nm (blue), from 500 to 600 nm (green), from 600 to 800 nm (red), and from 800 to 1,200 nm (NIR). Using a single scan, an RGB color image and an NIR image of the fluorescent cell sample were successfully captured with tens of photon signals in a 40 ms exposure time for each pixel. This result demonstrates that TES is a useful wide-band spectral photon detector in the field of life sciences.

Highlights

Spectral imaging provides information about the distribution of multiple molecules and other entities via spectral analysis
We introduce an improvement in confocal laser scanning microscopy (CLSM) optics to excite a wider range of fluorescent dyes and present the results of an extended investigation of cell samples
Color images of the bovine pulmonary artery endothelial cells (BPAECs) were successfully constructed from the datasets of red for mitochondria, green for F-acting, and blue for nuclei (Figure 2A)

Summary

Introduction

Spectral imaging provides information about the distribution of multiple molecules and other entities via spectral analysis. In the field of life sciences, spectral confocal laser scanning microscopy (CLSM) provides substantial information on the cellular dynamics associated with various biomolecules. In CLSM cell-imaging applications, samples are labeled with fluorescent dyes and irradiated by excitation light to obtain fluorescence signals. In living cells, excitation light irradiation deteriorates cells from their native state and causes photobleaching of fluorescent dyes (Icha et al, 2017). It is ideal to minimize excitation light irradiation. A decrease in the excitation light results in a decrease in the fluorescence signal. In the case of autofluorescence, which is emitted from endogenous fluorescent molecules in living organisms, signals are weak even when the molecules are irradiated with high-power excitation light

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