Name: Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy (Video) | JoVE Journal
Uploaded: 2025-08-29T13:50:00.000+00:00
Duration: 9 min 19 s
Description: Learn SLAM microscopy for label-free multimodal imaging of biological tissues using nonlinear light interactions. This method enables simultaneous four-channel detection to study cellular structures and disease mechanisms without toxicity.

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Funding for the equipment used in this study was provided in part by GSK through the Center for Optical Molecular Imaging at the Beckman Institute for Advanced Science and Technology. This work was also supported in part by the NIH/NIBIB Center for Label-free Imaging and Multiscale Biophotonics (CLIMB) (P41EB031772) and the NSF Science and Technology Center for Quantitative Cell Biology. K.K.D.T was supported by the NIEHS Research Training Program in Toxicology and Environmental Health (5T32ES007326-25), A.D.C was supported by the Beckman Institute for Advanced Science and Technology Postdoctoral Fellows program, and A.H. was supported by the NIBIB Tissue Microenvironment Training Program (T32EB019944). The authors thank Darold Spillman for his management of lab spaces, equipment, and technology and thank Drs. Janet E. Sorrells and Rishyashring R. Iyer for their past contributions to the SLAM microscope system used in this study.

The authors declare no conflicts of interest.

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...

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 intensity1,2,3. 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 specimen1,2,3. While traditional microscopy techniques involving a linear interaction with the illumination light also enable quantitative imaging, nonlinear microscopy permits a more multimodal approach, often having improved imaging depth and optical sectioning, while also causing less photodamage to the sample4. In the context of biomedical applications, such as intraoperative histology-like evaluation of tissue5,6, this technique enables the direct probing of native biomolecules, facilitating seamless investigations of living cells without the need for external markers or modifications. This approach avoids the use of tags, labels7, exogenous molecules, genetically expressed fluorescent proteins, or nanoparticles, which not only require extensive labor but also have the potential to disrupt the specimen's native biochemistry and negatively impact target cells or tissues8,9,10. Thus, the nonlinear microscope serves as a powerful and genuine analytical tool that provides continuous imaging of microscopic structures without altering the specimen's chemical and structural integrity. This capability opens a wide array of applications, ranging from diagnosis and drug discovery to cell signaling and systems biology.
Multiphoton contrasts such as two-photon autofluorescence (2PAF), three-photon autofluorescence (3PAF), second-harmonic generation (SHG), and third-harmonic generation (THG) each confer analytical advantages in characterizing samples. 2PAF is generated by endogenous flavins, with flavin adenosine dinucleotide (FAD) being the most abundant in biological systems11. 3PAF leverages near-infrared excitation to visualize intrinsic chromophores such as NAD(P)H and enables metabolic imaging while circumventing UV-related phototoxicity and attenuation issues12. SHG specifically reveals non-centrosymmetric molecular structures such as fibrillar collagen (types I, II, and III) and protein arrays13,14,15,16,17, providing structural insights1. Lastly, THG, sensitive to interfaces and small structural features, excels in imaging lipid distributions and extracellular vesicles18,19,20,21,22.
Each of these contrast signals has been independently explored by research groups, establishing the niche for multiphoton nonlinear microscopy as a reliable and versatile tool for biological applications23,24,25,26. These efforts were spearheaded by the inventors of the 2PAF microscope, who harnessed their innovative technology to make new insights into tissue morphology, cell metabolism, and even the morphofunctional features of pathologies such as Alzheimer's disease and cancer23. Their work has ultimately driven groundbreaking applications in neuroscience27,28. In the following years, several researchers highlighted the sensitivity of the SHG to changes in the extracellular matrix, the scaffold that provides structural support to cells and which undergoes substantial alterations during cancer pathogenesis29. Notably, Campagnola et al.14,24,30,31 demonstrated that SHG microscopy enables the visualization of macromolecular and supramolecular assemblies, revealing details such as the arrangement, concentration and type of collagen that are not detectable with conventional fluorescence-based techniques. These results underscored the potential of SHG microscopy as a viable diagnostics tool in clinical settings. Finally, THG microscopy was successfully applied to visualize retinal layers in rodents at subcellular resolution25, map brain structures32, and capture the multi-interfacial topography of melanoma cell invasion33.
These historical implementations of nonlinear microscopy generally involved a mode-locked femtosecond (fs) laser coupled to a high numerical aperture (NA) objective to generate and collect the nonlinear signals, which were then directed with a dichroic mirror to a bandpass filter and collected with a photomultiplier tube (PMT)34. Despite similar implementations, most applications focused on acquiring only one, or, at most, two contrasts, while overlooking the others. To fully realize the potential of the multiphoton microscope, Boppart et al. first implemented the sequential acquisition of multiple nonlinear contrasts21,35,36,37, followed by their simultaneous detection4,22,38,39,40,41. The resulting technique, known as Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy, spatially and temporally co-registers the nonlinear 2PAF, 3PAF, SHG, and THG signals through single-shot excitation and multiplex detection, thereby achieving superior analytical power. The spatiotemporal co-localization of these four channels provides highly ranked and feature-rich data42 offering advantages over sequentially acquired data by eliminating motion artifacts and ensuring improved image registration. Consequently, SLAM enables the generation of composite features, such as optical reduction-oxidation ratios, that effectively characterize metabolic properties43,44,45. Moreover, the combined analysis of signals across modalities facilitates the engineering of additional features based on the correlation and colocalization of channels. For instance, Shi et al. demonstrated the importance of the colocalization features of SLAM microscopy in accurately classifying Chinese Hamster Ovary (CHO) cells used in pharmaceutical production42. In this way, SLAM lays the groundwork for what is now considered the state-of-the-art in multiphoton microscopy, paving the way for future advancements in the field46.
In this protocol, we outline the key components of a SLAM microscope (Figure 1) and describe a step-by-step methodology for its implementation. This includes the generation and shaping of the excitation pulses, representative tissue specimen preparation, imaging procedures, and the processing, as well as the display of the resulting data. By utilizing off-the-shelf technology and established techniques, this protocol is universal, transferable, and can be easily reproduced using similar optical systems across different laboratories.
<img alt="SLAM microscope diagram; optical setup for scanning, excitation; photon detectors for spectral analysis." class="xfigimg" src="/files/ftp_upload/68637/68637fig1v2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: The optical design of a simultaneous label-free autofluorescence multiharmonic (SLAM) microscope. The femtosecond laser outputs 1040 nm light in 370 fs pulses at a repetition rate of 10 MHz. Intensity and polarization of the laser are carefully controlled before pumping a photonic crystal to generate a supercontinuum. The pulses are then compressed in by a pulse shaper and directed into a customized laser scanning microscope (dashed box). Dichroic mirrors and bandpass filters isolate the nonlinear signals generated by the sample before they are detected by high-sensitivity photon detectors such as photomultiplier tubes (PMTs) or hybrid photodetectors (HPDs). Optical design is based on a previously described system41. <a href="https://www.jove.com/files/ftp_upload/68637/68637fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

simultaneous label-free autofluorescence multi-harmonic microscopy

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

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.

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</st...

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.

Nonlinear optical microscopy images biological samples by detecting signals from the nonlinear interaction of ultrashort laser pulses with endogenous ...

Simultaneous label-free autofluorescence multi-harmonic microscopy, or SLAM microscopy, captures unique information in biological samples by measuring non-linear interactions with light. In our lab, we use this technique to study life and disease at the microscopic scale. We observe a shift toward label-free multimodal imaging, offering richer insights into cellular and tissue structures and dynamics, while avoiding toxicity and enabling complimentary contrasts and correlations across the modalities.A state-of-the-art label-free nonlinear microscopes acquire one signal at a time, risking misalignment, photo damage and a lot of concurrent processes. The SLAM overcomes these issues by enabling a strictly coregistered multimodal contrast, ensuring reliable spatial and temporal data. This technique enables simultaneous excitation and detection of four channels in a multiphoton microscope.Using photonic crystal fiber to broaden pulses for SHG, THG, and two-and three-photon fluorescence. SLAM captures multiplexed metabolic structural and dynamic cell signaling information without alignment artifacts or exogenous labels, enabling standardized quantitative tissue analysis. Its applications span fundamental biology, translational research, and biomarker discovery in oncology, neurodegeneration, and tissue regeneration.To begin, use a spectrometer or optical spectrum analyzer to measure the laser after the parabolic mirror and verify the supercontinuum generation of the photonic crystal fiber. Rotate the halfway plate after the polarizing beam splitter to optimize the width of the supercontinuum. Turn on the microscopy autocorrelator.In the software, set the detector to the internal detector and adjust the Scan Range to 5 picoseconds to capture the full width of the pulse autocorrelation. Unblock the laser and align the autocorrelator to the beam exiting the pulse shaper until a signal appears. Ensure that shutter 2 is closed.Then apply the appropriate immersion media, such as water or oil, to the objective lens. Place the autocorrelator external detector on the microscope stage and position it directly above the objective. Now turn on the scanning galvanometer mirrors to set their voltage to 0.Open the shutter and use a fluorescent target to confirm that the beam reaches the external detector. Set the autocorrelator detector to be the external detector. Then adjust the stage position until the signal appears.Adjust the polynomial coefficients of the pulse shaper to iteratively minimize the pulse width measured by the autocorrelator. As the pulse width decreases, reduce the scan range accordingly to improve the accuracy and visualization of the autocorrelation function. Ensure that the laser input to the photonic crystal fiber is blocked and turn on the laser.Allow the laser to warm up for 30 minutes. Verify that the beam path is free of obstructions, then place an optical power meter in the beam path just before the photonic crystal fiber. Press the Control button to open shutter 1 and measure the input power.Close shutter 1 again. Move the power meter to the output of the photonic crystal fiber after the parabolic mirror. Open shutter 1 and use the input three-axis stage to maximize the output power.Let the photonic crystal fiber warm up for 10 minutes then maximize the output power again using the three-axis stage and record the transmission of the PCF. Move the power meter after the neutral density filter and rotate the filter to adjust the laser power reaching the sample. Multiply the measured power by the transmittance of the remaining optics to determine sample power.Now, turn on the galvanometers and stage. Launch the acquisition software and enter the desired imaging parameters. Place the sample on the stage using the same immersion media that was applied earlier to the objective lens.Then turn off the room lights and close the curtains to prevent ambient light from entering. Next, switch on the detector power supplies and amplifiers. Shut the light box surrounding the microscope stage.Use the electronic control switches to manually open the detector shutters 3-6. In the acquisition software, press the Click to Start Acquisition option to begin SLAM imaging. Adjust the microscope focus using the stage controller knob until the sample is clearly visible.Use the joystick on the microscope stage controller to find the desired field of view. Once positioned, click Stop and Reset. Under the Saving tab, toggle Save Data to Yes.Then press on the Click to Start Acquisition option and wait for the imaging to complete. To manipulate the sample during imaging, close the detector shutters and use a flashlight or headlamp for illumination. After adjustments, return to the imaging setup.To end image acquisition, close the detector shutters, turn off the detectors, and only then switch on the room lights. Block the laser using shutter 1, then turn off the laser. Turn off the galvanometers and stage and close the acquisition software.Dispose of the tissue sample appropriately and clean the workstation. A properly cleaved photonic crystal fiber showed a flat end face perpendicular to its axis and a clean face. Poorly cleaved photonic crystal fibers showed angled cuts, chipped faces, and pronounced scratches or debris on the surface.Improper handling or contamination resulted in visible dirt and particulate buildup around the fiber surface obscuring the air hole pattern. A burned core with darkened regions indicated a damaged photonic crystal fiber at the end of its life. The supercontinuum spectrum spanned approximately 975-1, 175 nanometers with a flat top and distinct spectral modulations.Pulse autocorrelation after compression showed a symmetrical profile with a narrow full-width half-maximum, indicating good pulse shaping. Properly functioning supercontinuum sources produced strong second and third harmonic signals. A narrowed supercontinuum resulted in loss of third harmonic signal intensity while retaining the second harmonic signal.Absence of pulse shaping drastically reduced all nonlinear signals, yielding a faint and low contrast image. Successful SLAM imaging of ex vivo kidney tissue showed distinct nephron structures with multiple strong optical signals, including third harmonic generation and NADPH fluorescence. Renal interstitial collagen fibers were identifiable via strong second harmonic generation signals.The redox ratio image indicated elevated aerobic mitochondrial metabolism in the proximal tubules of the renal cortex compared to the medulla. A second redox ratio map confirmed spatial variation in oxidative and glycolytic metabolism, reinforcing differential energy usage within the tissue.

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Learn SLAM microscopy for label-free multimodal imaging of biological tissues using nonlinear light interactions. This method enables simultaneous four-channel detection to study cellular structures and disease mechanisms without toxicity.

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

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