Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

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 extraction^2,3, 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

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........

Materials

Name	Company	Catalog Number	Comments
Agarose	Wako Chemicals	312-01193
Beam splitters	Carl Zeiss, Nikon		MBSInVis405, MBS458, MBS488, MBS458/514, MBS488/543, or MBS 488/543/633 beam splitters (Carl Zeiss)
Confocal microscope	Carl Zeiss, Nikon		Model LSM 880 (Carl Zeiss), Model A1R (Nikon)
Cover slips	Matsunami Glass	C024601
Glass slides	Matsunami Glass	S011120
Half-reflection mirror	Carl Zeiss, Nikon		NT80/20
Laser power meter	Thorlabs		PM400 (power meter console) and S175C (sensor)
LB Broth	Nacalai tesque	20066-95	For bacteria culture
Image analysis software	The MathWorks		MATLAB version 2019a or later, Image Processing Toolbox is needed
Microscope objective	Carl Zeiss, Nikon	440762-9904	e.g. 63x plan Apochomat NA = 1.4 (Carl Zeiss)
Microscope software	Carl Zeiss, Nikon		ZEN (Carl Zeiss),NIS-elements (Nikon)
PBS(-)	Wako Chemicals	166-23555
Programming language			Python and libraries, modules (numpy, scikit-learn, scikit-image, os, glob, matplotlib, tkinter) are rquired to run the supplied PCA script.
Silicone gasket	ThermoFisher Scientific	P24744
Workstation			A high-performance workstation with discrete GPUs is recommended.
Yeast extract-peptone-dextrose (YPD) agar medium	Sigma-Aldrich	Y1500-250G	For yeast culture
YPD medium	Sigma-Aldrich	Y1375-250G