Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection

Summary

This protocol details the advanced imaging applications, and shared-resource management of a laser-scanning confocal microscope system integrated with a multiplexed array detector for high-resolution cellular and subcellular imaging. The workflow enables multimodal imaging from confocal to super-resolution (~120 nm) within a single platform, making nanoscale visualization more accessible.

Abstract

Super-resolution microscopy (SRM) overcomes the diffraction limit of conventional light microscopy, enabling nanoscale visualization of subcellular structures. However, the widespread adoption of techniques such as stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM) / stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) can be hindered by factors such as high phototoxicity, specialized sample preparation, or complex operation. Here, we present a comprehensive protocol that leverages a laser-scanning confocal platform integrated with a multiplexed super-resolution detector. This system, combined with computational processing, enables a straightforward transition between confocal and super-resolution imaging within the same workflow. The protocol provides a user-friendly approach for fast, high-resolution, and low-phototoxicity imaging. The detailed method encompasses multimodal imaging capabilities, including multicolor fluorescence, differential interference contrast (DIC), three-dimensional Z-stack reconstruction, time-lapse live-cell imaging, large-area tiling, and various super-resolution modes offered by the multiplexed array detector. Systematic comparisons demonstrate that the super-resolution modes achieve a lateral resolution of approximately 120 nm, representing about a two-fold improvement over the diffraction limit of conventional wide-field microscopy and a significant enhancement in both resolution and signal-to-noise ratio (4-8 fold) compared to standard confocal operation on the same platform. The discussion also covers key points of a shared resource management model essential for supporting the sustainable operation of this technology. This integrated guide serves as a valuable resource for both new and experienced researchers and facility managers aiming to adopt and maximize the potential of this accessible form of advanced imaging in life sciences research.

Introduction

The visualization of dynamic subcellular structures is fundamental to advancing life sciences research. However, the resolution of traditional optical microscopy is limited by diffraction to approximately 200 nm laterally, restricting its ability to resolve dynamic subcellular processes. To overcome this barrier, various super-resolution techniques have been developed. However, techniques such as STED microscopy, PALM/ STORM, and SIM often present barriers to widespread adoption in multi-user environments, including high phototoxicity, the need for specialized fluorophores or sample preparation, complex alignment procedures, or intensive computational processing

Protocol

All experiments involving biological samples were conducted in accordance with the guidelines and regulations of Core Facilities, Zhejiang University School of Medicine and were approved by the Institutional Biosafety Committee (Approval Certificate No. BSL20235710079).

NOTE: All procedures involving cell cultures must be performed under sterile conditions in a Class II biological safety cabinet unless otherwise specified. This protocol assumes the microscope system is installed, and laser powers are calibrated. Detailed specifications and recommended vendors for key reagents, consumables, and equipment (e.g., imaging dishes, cover glass, m....

Results

Multicolor fluorescence imaging
Multicolor fluorescence imaging of fixed COS7 cells was performed as described in Section 3. Antibodies against α-tubulin (microtubules) and TOMM20 (mitochondria) were used with DAPI nuclear counterstain. As shown in Figure 1, nuclei (blue), mitochondria (green, Alexa Fluor 488), and microtubules (red, Alexa Fluor 568) were clearly delineated with high specificity and minimal spectral crosstalk, validating the effectiveness of the sequenti.......

Discussion

The integrated protocol presented here provides a comprehensive roadmap for implementing the advanced imaging capabilities of a modern confocal microscope system equipped with a multiplexed array super-resolution detector, within a sustainable shared-resource framework. This section synthesizes the method's critical aspects, practical implementation, and its position in the bioimaging field. Successful execution of this protocol hinges on several pivotal steps. First, meticulous sample preparation is foundational; th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
COS7 cells	ATCC (or similar)	CRL-1651	African green monkey kidney fibroblast cell line.Used for preparation of fixed-cell samples for multicolor super-resolution imaging
Donkey anti-mouse IgG, Alexa Fluor 568 conjugate	Invitrogen (or similar)	A10037	Secondary antibody for microtubule staining. Used at 1:500 dilution
Dulbecco‘s modified eagle medium (DMEM), high glucose	Gibco (or similar)	11965092	Standard culture medium for COS7 cells, supplemented with 10% FBS.
Fetal Bovine Serum (FBS), heat-inactivated	Gibco (or similar)	10082147	Serum supplement used at 10% (v/v) in all cell culture media.
Glass-bottom Imaging Dish (35 mm, µ-Dish, #1.5 polymer coverslip, 0.17 mm thickness)	ibidi (or similar)	81156	Substrate for cell plating and imaging. Critical for compatibility with high-NA oil immersion objectives.
Image processing & analysis software (ZEN modules)	Carl Zeiss AG	ZEN 3.5 (blue)	Used for 3D reconstruction, super-resolution processing (e.g., Airyscan Processing), PSF measurement, and SNR analysis.
ImageJ / Fiji	Open Source	N/A	Used for additional 3D reconstruction, generating orthogonal views, and general image analysis.
Immersion oil (Refractive Index = 1.518)	Carl Zeiss (or similar)	444960-0000-000	For use with oil immersion objectives (e.g., 63x/1.4 NA).
Laser-scanning confocal microscope system with multiplex array detector (e.g., ZEISS LSM 900 with Airyscan 2)	Carl Zeiss AG	LSM 900 (Airyscan 2)	The primary imaging platform used for all confocal and super-resolution modes described in the protocol.
Minimum Essential Medium (MEM) with Earle‘s Salts	Gibco (or similar)	11095080	Standard culture medium for Hep3B cells, supplemented with 10% FBS.
Mouse anti-α-tubulin monoclonal antibody (Clone DM1A)	Sigma-Aldrich (or similar)	T6199	Primary antibody for labeling microtubules. Used at 1:500 dilution
Paraformaldehyde (PFA), 4% in PBS	Jinpan (or similar)	TBS5083	Fixative for cells. Use at room temperature for 15 min. Handle with appropriate precautions.
Phenol-red-free Imaging Medium	Gibco (or similar)	21063029	Low-autofluorescence medium used for live-cell imaging. Pre-equilibrated with 5% CO2 at 37°C prior to use.
Phosphate-buffered saline (PBS), 1X, pH 7.4	CellWorld (or similar)	C0162-311	Used as washing and dilution buffer throughout the protocol.
ProLong gold antifade mountant	Invitrogen (or similar)	P36930	Hardened mounting medium for preserving fluorescence in fixed samples.
Triton X-100	Sigma-Aldrich (or similar)	T8787	Used at 0.1% (v/v) for cell permeabilization
Tween-20	Sigma-Aldrich (or similar)	P9416	Used at 0.05% (v/v) in PBS (PBST) for washing steps after antibody incubations.
Bovine serum albumin (BSA), Fraction V	Sigma-Aldrich (or similar)	A7906	Used at 1% (w/v) in PBS for blocking and as a component of antibody dilution buffer.
DAPI (4‘,6-diamidino-2-phenylindole)	Sigma-Aldrich (or similar)	D9542	Nuclear counterstain. Used at a final concentration of 1 µg/mL.
Donkey anti-rabbit IgG, Alexa Fluor 488 conjugate	Invitrogen (or similar)	A21206	Secondary antibody for mitochondrial staining. Used at 1:500 dilution
Fluorescent microspheres (Beads), 100 nm diameter	Invitrogen (e.g., TetraSpeck™) or similar	T7279	Used for empirical measurement of the system‘s point spread function (PSF) and resolution.
Hep3B cells stably expressing EGFP-mitochondria	IMMOCELL	IM-H367	Human hepatoma cell line engineered to stably express EGFP targeted to the mitochondrial matrix. Used for live-cell time-lapse imaging.
HEPES buffer solution, 1 M	Gibco (or similar)	15630080	Used at a final concentration of 25 mM in imaging medium for pH stabilization during live imaging outside a CO2 incubator.
High-precision coverglass (#1.5, 0.17 mm thickness)	Marienfeld (or similar)	107052	Used for mounting fixed samples.
Microscope operating software (e.g., ZEN blue edition)	Carl Zeiss AG	ZEN 3.5 (blue)	Used for system control, image acquisition, and basic processing.
Objective lens: Plan-apochromat 63x/1.40 NA Oil DIC	Carl Zeiss AG	421462-9900-000	Primary objective used for high-resolution imaging.
Rabbit anti-TOMM20 polyclonal antibody	Abcam (or similar)	ab186735	Primary antibody for labeling mitochondria. Used at 1:1000 dilution