Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Summary

This protocol presents a step-by-step guide for the Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopic technique, including details on how to generate the laser light source, prepare a tissue sample, conduct imaging, and analyze the data. SLAM advances nonlinear microscopy by measuring four complementary label-free contrasts to investigate the tissue microenvironment.

Abstract

Nonlinear optical microscopy images biological samples by detecting signals from the nonlinear interaction of ultrashort laser pulses with endogenous molecules. This method allows fast chemical and structural identification at subcellular resolution in a label or tag-free and nondestructive manner, thereby enabling a powerful approach to investigate cells and tissues. These distinctive nonlinear contrasts include multiphoton-excited autofluorescence and harmonic generation. Because each of these contrasts offers unique advantages and limitations, their combination and their spatiotemporal co-registration provide a complementary contrast palette that enhances the analytical capabilities of nonlinear optical microscopy. Therefore, our group developed Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy, an imaging technique that measures four or more concurrently generated nonlinear optical signals, aiming to identify distinct morphological, metabolic, and functional features in biological specimens. Here, we present a protocol for SLAM imaging of tissues, focusing on essential components of the technique, including the laser source, pulse compression, and microscope. In addition, we discuss sample preparation and outline the data processing pipeline for SLAM data. The presented workflow is suitable for investigating the metabolic state, arrangement, cellular responses, and composition of both human and animal tissues without relying on exogenous labels.

Introduction

Nonlinear optical microscopy leverages the response of a specimen to intense optical radiation (light with electric fields larger than 3 × 10³ V/cm) to derive image contrast, where the induced polarization (and thus the signal generated) depends nonlinearly on the incident intensity^1,2,3. Because these multiple contrasts are each mediated by different light-matter interactions, nonlinear microscopy provides simultaneous access to unique information regarding both the morphology and chemical landscape of a specimen^1,

Protocol

All animal studies were performed as per the protocol UIUC#23031 approved by the University of Illinois Institutional Animal Care and Use Committee and strictly followed all relevant guidelines and regulations.

1. Cleaving and aligning photonic crystal fiber (PCF)

1. Using fiber cutters or wire cutters, cut a section of photonic crystal fiber (PCF, Table of Materials) to a length of slightly over 15 cm.
   NOTE: Lengths of at least 1.5 cm will be cleaved from each end of the PCF since this length is required to securely clamp the PCF during cleaving. The final length of the PCF (12 cm) used in th....

Results

The complex multichannel, multimodal approach of the SLAM microscope (Figure 1) can make it difficult for new users to troubleshoot issues and understand results. First, a well-cleaved PCF features a clean face (Figure 2C) with a flat surface that is perpendicular to the PCF's length (Figure 2A). An angled, chipped cleave face (Figure 2B) and the presence of scratches (Figure 2E

Discussion

Generating a suitable supercontinuum is crucial for successful SLAM imaging, but it can be challenging due to the complexity of setting up the PCF. Ensuring precise cleaving and careful handling of the PCF is essential to maintain clean surfaces free from debris, dirt, or scratches, all of which can interfere with effective coupling, supercontinuum generation, and even shorten the lifespan of the PCF. Since the PCF requires regular replacement, practicing fiber cleaving and fine-tuning cleaver settings can help achieve o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-axis stage	ThorLabs	MAX316	Discontinued.
35 mm glass bottom imaging dish	MatTek	P35G-0-14-C	Uncoated #0 coverglass.
DMEM	Gibco	21063029	High glucose, HEPES, no phenol red.
Endothelial cell growth medium supplements	PromoCell	C-39210	Omit Fetal Calf Serum.
FemtoTrain femtosecond laser	Spectra-Physics	1040-5	Average Power: >5.0 W, Pulse Energy: >500 nJ, Wavelength: 1040 nm ±8 nm, Repetition Rate: 10 MHz, Pulse Width (FWHM): <220 fs.
Fetal Bovine Serum	Cytiva	SH30071.03	HyClone Characterized.
Fiber stripper	ThorLabs	T10S13
Flavin adenine dinucleotide disodium salt hydrate	Sigma-Aldrich	F6625
Galvanometer mirrors	Cambridge Technology	6215H	Discontinued.
Glass processing workstation	Vytran	GPX3800
ImageJ	Eliceiri lab at the University of Wisconsin Madison	Version 2.16.0	FIJI Distribution.
Kimwipe tissue paper	Kimberly-Clark Professional	34120
L-Glutamine	Cytiva	SH30034.01
Microscope stage	Applied Scientific Instrumentation	FTP-2050
Microscope stage controller	Applied Scientific Instrumentation	MS-2000-500-UDSW
Microscopy autocorrelator	Angewandte Physik und Elektronik GmbH	Carpe
Objective lens	Olympus	XLPLN25XWMP2	Water immersion, 25X magnification, 1.05 NA.
Optical power meter	Newport	919P-003-10	600 - 1700 nm.
Optical spectrum analyzer	ThorLabs	OSA202C
Photonic crystal fiber	NKT Photonics	LMA-PM-15 NKT	Large mode area, polarization maintaining. Core Diameter: 15 µm.
Precision optical fiber cleaver	Fujikura	CT-101
Pulse shaper	Biophotonics Solutions Inc.	FemtoJock-D	Discontinued.
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate	Sigma-Aldrich	10128023001