Name: Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection (Video) | JoVE Journal
Uploaded: 2026-02-24T14:10:00.000+00:00
Duration: 7 min 42 s
Description: Learn super-resolution imaging for cellular biology using confocal microscopy with multiplex detection. This protocol enables ~120 nm resolution imaging of fixed and live cells with reduced phototoxicity.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors acknowledge the technical support from the Core Facilities, Zhejiang University School of Medicine and thank Yao Yao Zhong for providing the human liver cancer live-cell Hep3B sample.This work was supported by the Natural Science Foundation of Zhejiang Province (LZ25H060002), the Experimental Technology Project of Zhejiang University (SYBJS202321), the Zhejiang Provincial Department of Education (Y202351321), and the Open Research Project of the Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs (202201).

<ol><li>Prakash, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolution+in+super-resolution+microscopy+-+definition%2C+trade-offs+and+perspectives%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Resolution in super-resolution microscopy - definition, trade-offs and perspectives.</a> Nat Rev Mol Cell Biol. 25, 677-682 (2024).</li>
<li>Schermelleh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+microscopy+demystified%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Super-resolution microscopy demystified.</a> Nat Cell Biol. 21, 72-84 (2019).</li>
<li>Bond, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technological+advances+in+super-resolution+microscopy+to+study+cellular+processes%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Technological advances in super-resolution microscopy to study cellular processes.</a> Mol Cell. 82, 315-332 (2022).</li>
<li>Huff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+new+2D+super-resolution+mode+for+ZEISS+Airyscan%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">The new 2D super-resolution mode for ZEISS Airyscan.</a> Nat Methods. 14, 1223(2017).</li>
<li>Wu, X., Hammer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ZEISS+Airyscan%3A+Optimizing+Usage+for+Fast+Gentle%2C+Super-Resolution+Imaging%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">ZEISS Airyscan: Optimizing Usage for Fast Gentle, Super-Resolution Imaging.</a> Methods Mol Biol. 2304, 111-130 (2021).</li>
<li>Yin, W., Liu, S. S., Zhao, Q. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+strategy+for+fast+super-resolution+imaging+parameters+based+on+LSM+900%5BTitle%5D)+AND+%22Chin+J+Cell+Biol%22%5BJournal%5D)">Optimization strategy for fast super-resolution imaging parameters based on LSM 900.</a> Chin J Cell Biol. 47, 1644-1655 (2025).</li>
<li>Moore, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Actin+cables+and+comet+tails+organize+mitochondrial+networks+in+mitosis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Actin cables and comet tails organize mitochondrial networks in mitosis.</a> Nature. 591, 659-664 (2021).</li>
<li>Ranjan, R., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+live+cell+imaging+of+subcellular+structures%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Super-resolution live cell imaging of subcellular structures.</a> J Vis Exp. (167), e61563(2021).</li>
<li>Harmer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Airyscan+super+resolution+microscopy+to+study+trypanosomatid+cell+biology%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Airyscan super resolution microscopy to study trypanosomatid cell biology.</a> Methods Mol Biol. 2116, 449-461 (2020).</li>
<li>Lin, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+functions+and+management+of+laser+scanning+confocal+microscopy+LSM880+with+Airyscan%5BTitle%5D)+AND+%22J+Chin+Electron+Microsc+Soc%22%5BJournal%5D)">Advanced functions and management of laser scanning confocal microscopy LSM880 with Airyscan.</a> J Chin Electron Microsc Soc. 38, 271-275 (2019).</li>
<li>Li, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+study+on+imaging+characteristics+and+applications+of+LSCM880+with+Airyscan+and+spinning+disk+confocal+microscopy%5BTitle%5D)+AND+%22Technol+Innov+Appl%22%5BJournal%5D)">Comparative study on imaging characteristics and applications of LSCM880 with Airyscan and spinning disk confocal microscopy.</a> Technol Innov Appl. 32, 4-8 (2020).</li>
<li>Huff, J., Bergter, A., Luebbers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+mode+for+the+LSM+9+series+with+Airyscan+2%3A+fast+and+gentle+confocal+super-resolution+in+large+volumes%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Multiplex mode for the LSM 9 series with Airyscan 2: fast and gentle confocal super-resolution in large volumes.</a> Nat Methods. 16, 1159-1163 (2019).</li>
<li>Chen, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depletion+of+Vasohibin+1+speeds+contraction+and+relaxation+in+failing+human+cardiomyocytes%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Depletion of Vasohibin 1 speeds contraction and relaxation in failing human cardiomyocytes.</a> Circ Res. 127, 14-27 (2020).</li>
<li>Sandin, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near+simultaneous+laser+scanning+confocal+and+atomic+force+microscopy+%28conpokal%29+on+live+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Near simultaneous laser scanning confocal and atomic force microscopy (conpokal) on live cells.</a> J Vis Exp. (162), e61433(2020).</li>
<li>Castagnini, D., Palma, K., Jara Wilde, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+Imaging+of+Proteus+mirabilis+biofilm+by+expansion+microscopy%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Super-resolution Imaging of Proteus mirabilis biofilm by expansion microscopy.</a> J Vis Exp. (221), e67932(2025).</li>
<li>Wolf, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+live-cell+super-resolution+imaging+of+mitochondrial+cristae+and+quantification+of+submitochondrial+membrane+potentials%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Method for live-cell super-resolution imaging of mitochondrial cristae and quantification of submitochondrial membrane potentials.</a> Methods Cell Biol. 155, 545-555 (2020).</li>
<li>Wu, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-infrared+perylene+carboximide+fluorophores+for+live-cell+super-resolution+imaging%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Near-infrared perylene carboximide fluorophores for live-cell super-resolution imaging.</a> J Am Chem Soc. 146, 7135-7139 (2024).</li>
<li>Pfanzelter, J., Mostowy, S., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Septins+suppress+the+release+of+vaccinia+virus+from+infected+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Septins suppress the release of vaccinia virus from infected cells.</a> J Cell Biol. 217, 2911-2929 (2018).</li>
<li>Kolossov, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Airyscan+super-resolution+microscopy+of+mitochondrial+morphology+and+dynamics+in+living+tumor+cells%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">Airyscan super-resolution microscopy of mitochondrial morphology and dynamics in living tumor cells.</a> Microsc Res Tech. 81, 115-128 (2018).</li>
<li>He, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+macrophages+monitor+and+remove+particles+from+urine+to+prevent+tubule+obstruction%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Renal macrophages monitor and remove particles from urine to prevent tubule obstruction.</a> Immunity. 57, 106-123 (2024).</li>
</ol>

The authors declare no competing financial or non-financial interests. The authors used DeepSeek for language polishing and formatting assistance during the preparation of this manuscript.

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&#39;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...

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 processing1,2,3.
In this context, confocal microscope systems equipped with a multiplexed array detector offer a compelling alternative by enabling super-resolution imaging through multi-pixel detection and computational reassignment, significantly lowering the technical threshold4. This approach leverages a 32-element gallium arsenide phosphide (GaAsP) detector array to collect more photons from each scanning point. Subsequent intelligent deconvolution algorithms reassign this information, effectively reducing the detection volume. This process achieves spatial resolutions of approximately 120 nm laterally and 350 nm axially, representing about a 2x improvement over the diffraction limit of wide-field microscopy and yielding a 4-8x increase in signal-to-noise ratio compared to standard confocal mode on the same instrument4,5,6. This unique combination of enhanced resolution, superior signal quality, and compatibility with common fluorescent labels and protocols constitutes its primary advantage for broad application.
Consequently, this form of accessible super-resolution microscopy has found broad application in life sciences research, enabling detailed studies of organelle morphology, cytoskeleton dynamics, virus-host interactions, and molecular co-localization in both fixed and living cells7,8. Its relative gentleness makes it particularly suitable for extended time-lapse observations of live samples.
The integration of such advanced systems into core facilities is crucial for democratizing access, but their sustainable and effective operation presents distinct challenges. In institutional core facilities, such as at the Zhejiang University School of Medicine, optimal performance and impact depend critically on three pillars: (1) standardized and reproducible imaging protocols, (2) rigorous routine maintenance, and (3) an effective management framework for shared access. While valuable resources exist for specific aspects, such as imaging methods for earlier systems9or general facility management principles10,11, a comprehensive guide that integrates detailed, reproducible protocols for modern multiplexed array detector platforms with a proven shared-resource management model is currently lacking. This protocol article aims to bridge this gap.
The overarching goal is to provide a comprehensive, step-by-step guide for effectively implementing and utilizing a confocal system with a multiplexed array detector for super-resolution imaging within a shared-resource setting. It is designed for two primary audiences: researchers requiring resolution beyond conventional confocal limits (~250&#8236;-300 nm) but for whom live-cell compatibility, ease of use, and throughput are also critical; and facility managers establishing or optimizing such a service. Herein, we systematically detail the system configuration, multimodal imaging protocols (covering multicolor, 3D, live-cell, tiling, and super-resolution modes), and a proven shared management model. This integrated guide serves as a resource to maximize the potential and accessibility of this powerful imaging modality.

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

super-resolution imaging and shared management: a protocol for confocal microscopy with multiplex detection

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

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.

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...

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.

Super-resolution microscopy (SRM) overcomes the diffraction limit of conventional light microscopy, enabling nanoscale visualization of subcellular ...

Our research focuses on developing accessible super-resolution imaging methods to visualize dynamic subcellular structures, overcoming the diffraction limit of conventional microscopy. The super-resolution method face phototoxicity, complex operation, and specialized preparation. This protocol reduces these barriers through confocal-based multiplex detection.To begin, power on the microscope main unit, the laser launch module, the LED light source, and the control computer. Launch the microscope operating software. From the main interface, select GFP, then choose turn the reflected light off.Navigate to the Acquisition panel. From the objective lens dropdown menu, select the appropriate objective lens. For live cell imaging, 45 to 60 minutes before imaging, activate the environmental chamber and the active focus stabilization system.After the system is stabilized, verify the stability using the chamber sensors. Apply immersion oil on the objective's front lens without introducing bubbles. Place the prepared sample on the microscope stage.Raise it carefully until the coverslip contacts the immersion oil. Next, open the acquisition software. Go to the Acquisition tab and select create a new experiment.Now, add imaging tracks for the fluorophores used. For each track, set the confocal pinhole diameter to one air unit. To optimize the laser power, select the first channel and switch to live scanning mode.Set the initial laser power to a low value, then set detector gain to a moderate level within its linear range. After that, increase the laser power gradually until the target structures are clearly visible above the background in the live image. Set the pixel size using the software's Nyquist sampling button to calculate it automatically.Next, set the pixel time. Now, select the sequential scan mode between tracks to minimize crosstalk. Click Snap to acquire a single image.Save the image with relevant metadata. For consistent settings across optical sections, confirm that all imaging parameters are configured on a single optimal focal plane before defining the Z-stack. Open the Acquisition tab and select the Z-stack mode.Define the volume of interest by using a fast speed live scan to focus first on the bottommost structure of interest, then click Set Last or Bottom. Carefully adjust focus up to the topmost structure of interest and click Set First or Top. The software will then display the total depth.The Z-stack size is set to meet the Nyquist sampling criterion. Use the Optimal button to calculate it automatically. After configuring the imaging parameters for each channel, increase laser power or gain slightly for weak signals compared to a single two-dimensional image.Start the acquisition to collect the entire image stack. For visualization, use the microscope's native processing module. Generate maximum intensity projections to view all in focus signals in a single two-dimensional image.Then perform volume rendering using the softwares built-in algorithms to generate a three-dimensional representation. Adjust the brightness, opacity, and lighting as needed. Subsequently, generate orthogonal XZ and YZ cross-sections to inspect axial extent and registration.Multicolor fluorescence imaging of fixed COS-7 cells showed that the nuclei, mitochondria, and microtubules were clearly labeled with minimal overlap, confirming effective sequential scanning and optimized detection. The maximum intensity projection integrates signals across all optical sections, highlighting the spatial distribution of fluorescently labeled structures. A three-dimensional reconstruction provides a depth aware view of the sample architecture.Time-lapse imaging of live Hep 3B cells with EGFP tagged mitochondria is compared across modes. Confocal frames across time points show clear structures, but gradual signal loss. Standard super resolution or SR frames show higher resolution, but increased photo-bleaching over time.Fast super resolution or SR-4Y images preserve stable fluorescence even at later time points. The graph of mean EGFP fluorescence intensity over 100 time points demonstrates that the SR-4Y mode maintained the most stable signal. Imaging performance in fixed COS-7 cells was compared across modes, and super-resolution modes provided clearer structural detail than confocal.Measuring the point spread function using 100 nanometer fluorescent beads showed that Airyscan SR with subsequent joint deconvolution improved both lateral and axial resolution compared to Airyscan SR alone. This protocol allows researchers to study organelle morphology, cytoskeleton dynamics, and molecule colocalization in both fixed and live cells at approximately 120 nanometer resolution. The most critical consideration of this protocol is meticulous sample preparation using high-quality cover glass and optimized staining to ensure signal integrity for super-resolution processing.Following this procedure, researchers can perform quantitative image analysis, such as measuring signal-to-noise ratio or colocalization, using dedicated the software like ImageJ.

super-resolution-imaging-shared-management-protocol-for-confocal

Research

JoVE Journal

Biology

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Conducting Multiple Imaging Modes with One Fluorescence Microscope

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image analysis technique.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Spectral Iterative Bleaching Extends Multiplexity (IBEX) builds upon the base IBEX technique by adding heparin blocking to minimize nonspecific binding and leveraging spectral detection with computational unmixing to suppress autofluorescence. This approach accelerates image acquisition while reducing sources of background, enabling robust multi-round, high-parameter spatial proteomic analyses.

High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

This protocol outlines the procedure for performing deep, whole-cell single-molecule localization microscopy (SMLM) using a spinning disk confocal microscope with optical photon reassignment, thereby enabling DNA-PAINT imaging without the need for custom optics or complex illumination.

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Learn super-resolution imaging for cellular biology using confocal microscopy with multiplex detection. This protocol enables ~120 nm resolution imaging of fixed and live cells with reduced phototoxicity.

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

Explore More Videos