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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals.

Abstract

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Introduction

Diverse biomolecules within a cell1 emit autofluorescence signals, and the innate fluorescence signature of a cell consists of the assembly of these signals. This signature fluorescence reflects various cellular properties and also differences in physiological status. Analysis of innate fluorescence is minimally invasive and can complement traditional, more invasive microbiological probes that leave a range of traces from mild metabolic modification to complete cell destruction. While traditional techniques such as DNA or cell content extraction2,3, fluorescent in situ hybridization

Protocol

1. Preparation of the sample

  1. Place a 1 mm thick silicone gasket with wells on a glass slide.
  2. Place a 1 mm thick 0.8% (w/v) agarose slab in the well of the silicone gasket.
  3. Dilute the cell density of an arbitrary microbial cell culture to an optical density at 600 nm (OD660) = 1.0.
  4. Place a 5 µL aliquot of cell suspension on the agarose slab.
  5. Cover gently with a glass coverslip.

2. Setup of a microscope

Representative Results

Figure 1A shows the typical single-cell fluorescence signature of a bacterial cell presented as a traditional spectrum plot (top) and as a heatmap (middle). Figure 1B shows the result of an accurate 2D cell segmentation superimposed over the original CRM image of a population of soil bacteria (Pseudomonas putida KT2440)12. The resulting innate fluorescence signatures for the population are presented as a heatmap in

Discussion

There are two critical points in this method that need to be closely followed to obtain reproducible results: 1) keep the laser power output under the microscope objective consistent through excitation wavelengths and experiments, and 2) perform accurate cell segmentation.

The first point is particularly important when comparing the innate fluorescence signature among different experiments. Avoid simply applying the same “percent output” settings to the excitation wavelengths (i.e........

Acknowledgements

This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, and Technology of Japan (18K04843) to Y. Yawata, the JST ERATO (JPMJER1502) to N. Nomura.

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Materials

NameCompanyCatalog NumberComments
AgaroseWako Chemicals312-01193
Beam splittersCarl Zeiss, NikonMBSInVis405, MBS458, MBS488, MBS458/514, MBS488/543, or MBS 488/543/633 beam splitters (Carl Zeiss)
Confocal microscopeCarl Zeiss, NikonModel LSM 880 (Carl Zeiss), Model A1R (Nikon)
Cover slipsMatsunami GlassC024601
Glass slidesMatsunami GlassS011120
Half-reflection mirrorCarl Zeiss, NikonNT80/20
Laser power meterThorlabsPM400 (power meter console) and S175C (sensor)
LB BrothNacalai tesque20066-95For bacteria culture
Image analysis softwareThe MathWorksMATLAB version 2019a or later, Image Processing Toolbox is needed
Microscope objectiveCarl Zeiss, Nikon440762-9904e.g. 63x plan Apochomat NA = 1.4 (Carl Zeiss)
Microscope softwareCarl Zeiss, NikonZEN (Carl Zeiss),NIS-elements (Nikon)
PBS(-)Wako Chemicals166-23555
Programming languagePython and libraries, modules (numpy, scikit-learn, scikit-image, os, glob, matplotlib, tkinter) are rquired to run the supplied PCA script.
Silicone gasketThermoFisher ScientificP24744
WorkstationA high-performance workstation with discrete GPUs is recommended.
Yeast extract-peptone-dextrose (YPD) agar mediumSigma-AldrichY1500-250GFor yeast culture
YPD mediumSigma-AldrichY1375-250G

References

  1. Monici, M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnology Annual Review. 11, 227-256 (2005).
  2. Tang, J. Microbial metabolomics. Current Genomics. 12, 391-403 (2011).
  3. Woo....

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