Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Summary

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

Abstract

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with ¹³C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol's standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

Introduction

Cellular metabolism stands as a foundational pillar in cell biology, steering many processes that determine cell health, function, and interaction with the environment. Analyzing metabolism at the individual cell level, particularly within the native environments, provides invaluable insights to reveal the heterogeneous and complex activities in biological systems¹. This is especially crucial in the study of microorganisms, as many microbes exhibit unique growth requirements or environmental dependencies that challenge traditional cultivation methods². For instance, some species may require specific nutrient compositions....

Protocol

The use of bacterial specimens in this study is in accordance with the guidelines of the Institutional Review Board (IRB) of Boston University and the National Institute of Health.

NOTE: The general workflow followed in this protocol is summarized in Figure 2.

1. Bacterial culture and isotope labeling (Figure 2A)

NOTE: The example given here is for labeling a pure bacterial culture. If applying this protocol to polymicrobial communities, the medium and the labeling time must be adjusted accordin....

Results

The general workflow for single-cell microbial metabolic analysis with genetic identification by OPTIR-FISH is summarized in Figure 2. The representative results demonstrating the single-cell metabolic imaging capability of OPTIR are shown in Figure 3. This example used E. coli cells incubated with ¹²C- or ¹³C-glucose for 24 h. The incorporation of ¹³C into proteins has been shown to cause a significant red shift of protein amide.......

Discussion

Here, we described a detailed protocol for applying the OPTIR-FISH platform for simultaneous identification of microbial species and quantification of metabolic activities at the single-cell resolution. The critical steps include culture with stable isotope labeling for studying specific metabolic activities and fluorescence in situ hybridization for identifying target microbial species. Multi-channel fluorescence imaging and OPTIR imaging at selected wavenumbers could be performed sequentially on the same micro.......

Materials

Name	Company	Catalog Number	Comments
96% Ethanol	ThermoScientific	T032021000
Calcium Fluoride	Crystran	CAFP10-0.35
D-(+)-Glucose	Sigma-Aldrich	G7021-1KG
D-Gluocose (U-13C6, 99%)	Cambridge Isotopic Laboratories	CLM-1396-1
Ethylenediaminetetraacetic acid	Sigma-Aldrich	E9884-100G
formamide	ThermoScientific	17899
Luria-Bertani broth	Sigma-Aldrich	L3522-250G
M9 Minimal Salts 5x	SIGMA	M6030-1KG
OPTIR instrument	Photothermal Spectroscopy Corp.	mIRage LS
Paraforaldehyde Solution, 4% in PBS	ThermoScientific	J19943-K2
poly-L-lysine solution 0.1% (w/v)	Sigma-Aldrich	P8920-500ML
Sodium Chloride	Sigma-Aldrich	S9888-25G
Sodium dodecyl sulfate	Sigma-Aldrich	L3771-25G
Tris(hydroxymethyl)aminomethane hydrochloride, 99+%	ThermoScientific	A11379.18
Trypic Soy Broth	Sigma-Aldrich	22092-500G