Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy

  1. Gert-Jan Bakker
  2. Sarah Weischer
  3. Júlia Ferrer Ortas
  4. Judith Heidelin
  5. Volker Andresen
  6. Marcus Beutler
  7. Emmanuel Beaurepaire
  8. Peter Friedl

  • Curated by eLife

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    Summary: Nonlinear microscopy is in the unique position that high-resolution images of cells and other tissue components can be obtained in live tissue. However, scattering and absorption limit the penetration depth. The impact of nonlinear microscopy in biomedicine and biology would be much improved if higher imaging depths can be achieved. In this manuscript, the authors show they can accomplish imaging in complex specimens using 3- and 4-photon excitation, deeper in the specimen than comparable optics can accomplish with 2-photon excitation laser scanning microscopy. Using a customised commercial system, the authors have incorporated a high-powered laser source with an OPA and dispersion compensation to generate either 1330nm or 1650nm laser lines with high peak pulse energies at low pulse repetition rates. They then compare the relative capabilities of each laser line in terms of number of fluorescence emission channels measured (skin tumour xenografts), fluorescence bleaching analysis and functional toxicity thresholds and fluorescence signal attenuation (excised murine bone).

    This is a very interesting study with some potentially important findings from a technical perspective. However, there is a disconnect at present between the quality of the work and the quality of the presentation. There are many areas of quantitative imaging and intravital imaging that are well known to those in the direct field, and that are a complete mystery to the vast majority of those that are not. It would therefore be highly beneficial to restructure the manuscript in such a way that the findings can reach the many researchers that could benefit from this powerful approach rather than the few who already use it.

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Abstract

Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs.

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  1. Version published to 10.7554/elife.63776 on eLife
  2. eLife

    Reviewer #3:

    In this paper the authors have developed a system to simultaneously generate two-, three- and four-photon fluorescence excitation from a single laser line and then proceed to apply this system to a number of turbid biological imaging applications to highlight its capabilities. Using a customised commercial La Vision BioTec Trimscope, they have incorporated a high powered fiber laser source with an Optical parametric amplifier and dispersion compensation to generate a either 1330nm or 1650nm laser lines with high peak pulse energies at low pulse repetition rates. They then compare the relative capabilities of each laser line in terms of number of fluorescence emission channels measured (skin tumour xenografts), fluorescence bleaching analysis and functional toxicity thresholds and fluorescence signal attenuation (excised murine bone).

    Whilst the paper is well written, the concept of utilising high laser peak powers and at low repetition rates to generate 3PE and 4PE at spectral excitations at 1300nm and ~1650nm is not new and has been presented previously (Cheng et al. 2014), as referenced by the authors. The authors have however gone into more detail and presented a number of comparative excitation approaches to compare and contrast low-duty-cycle high pulse-energy infrared with the more common high-duty-cycle low pulse energy near-infrared alternative. The benefits of higher order multiphoton microscopy when combined with higher wavelength excitation allows deeper imaging and more localised fluorescence excitation with reduced phototoxic and photobleaching effects per excitation pulse. One of the major issues associated with generating 4PE is that since higher pulse energy is required, this further reduces the repetition rate of the laser source, in order to reduce the average laser power in order to avoid sample heating effects. This in turn leads to much longer acquisitions and is limited by the fluorophore saturation particularly since they are using single beam excitation.

    Major comments:

    1. It seems as though when you take into consideration duty cycles, fluorescence saturation, water absorption effects and longer acquisition times, which lead to greater phototoxicity, 4-PE at 1700nm excitation is not appropriate for most dynamic biological applications where acquisition speed and/or continued image acquisitions are the key factors. Could the authors comment on this?

    2. How long does it take to acquire a single frame with four-photon excitation at 1700nm? In none of the data sets was frame time mentioned in particular when acquired 3D data sets. Can the authors ensure that these times are mentioned both in the main text and the figures containing images.

    3. In line 131 and figure 3d the authors present data showing relative axial resolution measurements. Are these features measured diffraction limited and how do they know? They are clearly not measuring like for like structures (different fluorescent species) so do not think this can be used as a measure of resolution. Can the author provide other resolution measurements?

    4. In line 140 - 142 the authors present data showing the advantages of THG at 1650nm over other excitation lines. Aside from the excitation wavelength could this data be explained by the greater absorption and scattering at the emission wavelengths generated at these laser lines?

    5. In figure 3A and 3C the SNR for 1650nm increases whilst for 1300nm and 1180 excitation this decreases. Is this simply due to more of the exciting fluorophore species residing deeper into the tissue?

  3. eLife

    Reviewer #2:

    Nonlinear microscopy is in the unique position that high-resolution images of cells and other tissue components can be obtained in live tissue. However, scattering and absorption limit the penetration depth. The impact of nonlinear microscopy in biomedicine and biology would be much improved if higher imaging depths can be achieved. Lately a few key studies have appeared achieving this. This manuscript contains a well-motivated extension of this research, in particular on the benefits of high-pulse-energy low-duty-cycle infrared excitation near 1300 and 1700 nm over 2-photon excitation, in heterogenous and dense tissue. The authors compare three types of excitation, at 1650 and 1300 nm at 1 MHz and at 1100 (or 1270) nm at 80MHz. They characterize photodamage in the tissue and determine the limits for power densities to stay below that. They study the achieved resolution at high depth for each of the processes and show a deeper imaging depth is resolved in bone and tumor core with 3P and 4P than with 2P. The article is a very solid and extensive study.

    Though I have no major concerns with article, I do have some minor points:

    l.57: Are the resolutions reported for 2-, 3-, or 4- photon processes. Do you not expect these to differ for the different processes? l.60 It is not explained that power is increased from X to Y, instead the peak power of 87 nJ in L 67 is not found back in fig. S2.

    L. 103 Given is the power at the sample surface, after which the readout for cell stress via Ca imaging is done (very elegant). Is not the imaging depth of the readout relevant too, as it is probably the power density at the focus which matters. What imaging depths can be reached with this low power? This comes back later, but would be good to mention here.

    L.110 The phrase 'Furthermore' confuses me. I guess the authors mean to say that with their 2.8-8.7 nJ of power they were well below the 100 mW level? Which is kind of obvious at 1 MHz?

    L. 126 Some words are missing, 'but 1180'.

    Why do some signals show a peak in intensity in fig. 3C and G rather than a slope?

  4. eLife

    Reviewer #1:

    In this manuscript, the authors show they can accomplish imaging in complex specimens using 3- and 4-photon excitation, deeper in the specimen than comparable optics can accomplish with 2-photon excitation laser scanning microscopy. This is a clear advantage for imaging optically hostile specimens such as cultured organoids or spheroids, or in challenging in vivo settings. I am excited about these findings, but I am not at all supportive of the current version of the manuscript being used to present these lovely findings.

    There are two strong reasons for my opinion:

    i. The manuscript presents the findings in a manner that will only be understandable by the readers who are familiar with the topic, and who are likely to already have heard of the capabilities of 3- and 4-photon excitation to image deeper into specimens.

    ii. The results are not presented in a way that the large body of potential readers can understand. They will be unable to grasp the way that the experiments were performed, or understand what the figures are showing, or critically evaluate the results that are presented.

    Thus, there is a disconnect between the quality of the work and the quality of the presentation. There are many areas of quantitative imaging and intravital imaging that are well known to those that know about them (or use them), and that are a complete mystery to the vast majority of those that don't know about the tools or use them. The authors must take this as an opportunity to reach the many workers that could benefit from this powerful approach, rather than writing for the group that already knows (and even uses) the approaches presented.

    1. Provide needed background and present important things first. The authors should give the reader a clear view into the issues in imaging biological tissues with the longer wavelengths that are used for confocal laser scanning microscopy (CLSM) and for two-photon laser scanning microscopy (TPLSM). There are several factoids presented, all seemingly true, but not presented in an accessible manner. Rather than starting with a mention of the expected temperature rise due to the dramatically higher absorbance by water of 1300nm and 1700nm light, the paper first presents the major absorbance of the light (~2/3 loss) and that this isn't a problem because there is sufficient laser power. For most readers, the need for a larger laser won't be their first question; instead it will be the viability after/during the imaging session. The expected temperature rise, and an indirect mention of burn marks (!), comes at the end of the section.

    2. Explain and perform cell viability tests. Calcium imaging for assessing tissue viability is not the technique of choice for most readers, and is presented in a way that assumes general knowledge that simply does not exist. Membrane patency assays using membrane-impermeant DNA dyes, or other live-dead assays are far more common, but not presented in this study. I am not insistent that the authors use any particular assay, but I am insistent that the authors present the need for viability assay(s), teach the reader the principles of the assay(s) used, and present the results in an understandable manner.

    3. Present the finding and the figures in an accessible manner. The figures are simply not digestible by the readers who do not perform this sort of work, and the legends do not help sufficiently. For those of us who do perform work of this sort, the figures are not as convincing as they should be, or presented in a way that they can be critically evaluated.

    Consider the legend for Figure 1: "Microscopy with simultaneous 2-, 3- and 4 photon processes excited in fluorescent skin tumor xenografts in vivo. Representative images were selected from median-filtered (1 pixel) z-stacks, which were taken in the center of fluorescent tumors through a dermis imaging window. a) Excitation at 1300nm (OPA) in day-10 tumor at 145 μm imaging depth with a calculated 3.3 nJ pulse energy at the sample surface, 24 μs pixel integration time and 0.36 μm pixel size. For calculation of pulse energy at the sample surface see Figure S3. b) Excitation at 1650 nm (OPA) in day-13 tumor at 30 μm depth with a calculated 6.3 nJ pulse energy at the sample surface, 12 μs pixel integration time and 0.46 μm pixel size. c) Excitation at 1650 nm (OPA) in day-14 tumor at 85 μm depth, with a calculated 5.4 nJ pulse energy at the sample surface, 12 μs pixel integration time and 0.46 μm pixel size. Cell nuclei containing a mixture of mCherry and Hoechst appear as green."

    If I gave any of the figures and legends to the people in my lab, the half that don't do multiphoton imaging (but that have sat through many lab meetings) would just hand them back to me with quizzical expressions on their faces.

    The figures are not as compelling as the results, and defer to the body of the paper to explain what was done or what was shown, and assumes that the average reader remembers the differences between OPO and OPA , for example (which they won't). The power plots showing nJ and mW in Figure 3 are inaccessible to most readers, and not well described.

    I should mention that the figures, legends and text are not satisfying for the readers who are familiar with 2-, 3- and 4-photon imaging either. These are fantastic findings, and deserve figures that are as lovely as the results, and are compelling. Some of these issues are due to typos: "Consistently, multiparameter recordings were achieved inside the tumor at 350 μm depth using excitation at 1650 nm and 1300 nm, but 1180 nm (Figure 3b). "

    However, the greater problem is that the text doesn't present the findings in a straightforward, convincing fashion and then interpret them. Instead, the conclusion often leads the evidence: "In line with an improved depth range, the signal-to-noise ratio (SNR) of 3PE TagRFP outperformed the SNR of 2PE TagRFP at depths beyond 150 μm (Figure 3c). Because H2B-eGFP expression in HT1080 tumors was very high, 3PE eGFP emission reached the highest SNR."

    The legend and figure that it describes should be able to stand on their own, and convince a skeptical reader with the help of the text in the body of the manuscript.

    In summary, these are lovely and important results that I am excited about. They are presented in a fashion that will make it difficult for most to appreciate because the body of the paper is not fashioned to teach the reader, and the figures themselves are challenging, and the legends inadequately present what is shown in the figures. Careful expansion and editing should resolve all of these issues and make the manuscript into the presentation these excellent findings deserve.

  5. eLife

    Summary: Nonlinear microscopy is in the unique position that high-resolution images of cells and other tissue components can be obtained in live tissue. However, scattering and absorption limit the penetration depth. The impact of nonlinear microscopy in biomedicine and biology would be much improved if higher imaging depths can be achieved. In this manuscript, the authors show they can accomplish imaging in complex specimens using 3- and 4-photon excitation, deeper in the specimen than comparable optics can accomplish with 2-photon excitation laser scanning microscopy. Using a customised commercial system, the authors have incorporated a high-powered laser source with an OPA and dispersion compensation to generate either 1330nm or 1650nm laser lines with high peak pulse energies at low pulse repetition rates. They then compare the relative capabilities of each laser line in terms of number of fluorescence emission channels measured (skin tumour xenografts), fluorescence bleaching analysis and functional toxicity thresholds and fluorescence signal attenuation (excised murine bone).

    This is a very interesting study with some potentially important findings from a technical perspective. However, there is a disconnect at present between the quality of the work and the quality of the presentation. There are many areas of quantitative imaging and intravital imaging that are well known to those in the direct field, and that are a complete mystery to the vast majority of those that are not. It would therefore be highly beneficial to restructure the manuscript in such a way that the findings can reach the many researchers that could benefit from this powerful approach rather than the few who already use it.

  6. Version published to 10.1101/2020.09.29.312827v1 on bioRxiv