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 or symbiotic relationships not easily replicated in a laboratory setting, thus rendering them non-culturable by standard techniques³. Furthermore, the extended periods needed for the cultivation of certain species pose significant challenges for microbiological research, often extending beyond practical time frames for study and analysis. To circumvent these limitations, alternative methods such as polymerase chain reaction and fluorescence in situ hybridization (FISH) allow for the identification of microbial species without necessitating cultivation⁴, which can achieve a more accurate and holistic view of microbial ecosystems. However, these analytical technologies lack the ability to elucidate cellular metabolism. This gap highlights the ongoing challenges in the microbiology field: the concurrent task of differentiating cellular identity and elucidating metabolism at the single-cell level. Advancements in techniques such as imaging mass spectrometry (IMS) coupled with stable isotopes have emerged as powerful tools for single-cell metabolic analysis⁵. In these experiments, cells were incubated with substrates containing isotopes such as ¹³C or ¹⁵N. The newly anabolized biomolecules carry these isotopes, making them distinguishable on the m/z spectra. However, IMS suffers from expensive instrumentation, complicated sample preparation, relatively low throughput, and expertise required for analyzing the m/z spectra. When combined with molecular marker-specific fluorescence imaging, advancements have been made in elucidating cellular metabolism with increased specificity⁶. Nevertheless, challenges persist in bridging these two modalities. The difference in resolution between IMS and fluorescence imaging, combined with different operational setups, makes it difficult to align and correlate findings⁷.

The integration of vibrational spectroscopic imaging with stable isotope labeling offers a novel solution for the study of single-cell metabolism. The incorporation of heavier isotopes slows down the vibration of chemical bonds, leading to red-shifted peaks in the vibrational spectra⁸. Notably, vibrational imaging provides a spatial resolution comparable to fluorescence imaging, and both metabolic quantification and cell identification can be performed in a single setup, simplifying the image registration and correlation. Our recent work has demonstrated the combination of an advanced vibrational imaging platform: optical photothermal infrared (OPTIR) with fluorescence in situ hybridization (FISH) to probe glucose metabolism in bacterial communities⁹ (Figure 1). OPTIR is a vibrational spectroscopic microscopy system that harnesses the photothermal effect of mid-IR absorption by detecting the visible light change, which provides sub-micrometer resolution as in optical microscopy but with the additional vibrational spectroscopic information originating from the mid-IR absorption. FISH is a commonly used technique to determine the microorganism identity at the single-cell level. The sequence of oligonucleotides could be designed to target specific 16S sequences of different taxa, and different fluorophores could be attached. Specific hybridization of the designed oligonucleotide probes with the target rRNA leads to strong fluorescence signals of target species within individual cells, and multi-channel fluorescence imaging could be performed to identify multiple species within the population. As both OPTIR and fluorescence imaging are based on optical detection, combining OPTIR and FISH fluorescence is straightforward to implement. The two modalities share the same optical resolution for single-cell analysis and can be switched conveniently without requiring additional alignment or co-registration.

This protocol presents a detailed guide to leveraging the OPTIR-FISH platform for advanced single-cell structure-function analysis. We used the bacterial samples grown in ¹³C-glucose-containing media as a test bed and quantified de novo protein synthesis from ¹³C-glucose. In order to demonstrate the ability of the platform to identify cells, we used bacterial mixtures in which each species was labeled using rRNA-targeted FISH probes. This approach facilitated precise single-cell identification of specific bacterial strains, such as Escherichia coli (E. coli) and Bacteroides thetaiotaomicron (B. theta), and their metabolism. This protocol offers researchers a powerful tool for simultaneous metabolic profiling and species identification at the single-cell level, promising to advance our understanding of cellular interactions, physiology, and their roles in complex environments.

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…

Representative 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

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