We thank R. Heintzmann for fruitful discussions during the development of the striped-illumination approach, K. Rajewsky and A. Haberman for providing C57BL/6 B1-8 GFP transgenic mice. We acknowledge the Deutsche Forschungsgemeinschaft under grant NI1167/3-1 (to R.N.), HA5354/4-1 and SFB633, project A15 (to A.E.H.), the Charit&#233; under grant Rahel-Hirsch fellowship (to R.N.) for financial support. We particularly acknowledge the network JIMI for fruitful discussions and infrastructural support

Multi-beam Setup for Striped-illumination TPLSM
The setup used here is a specialized multi-beam two-photon laser-scanning microscope, as described previously6,20. The system is illustrated in Figure 1. The approach can be applied to other two-photon laser-scanning microscopes, which are able to synchronize camera acquisition with the movement of the galvoscanner mirrors, even if they are only capable to perform single-beam scanning. In this case, the disadvantage will...

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Volker Andresen is a shareholder of LaVision Biotec GmbH, Bielefeld, Germany. The other authors do not have&#160;any conflicts of interest.

The aim of intravital optical imaging is to dynamically and functionally visualize cellular motility and interactions in order to understand tissue and organ function in health and disease5. The most powerful technology to achieve this, multi-photon laser-scanning microscopy, still has to overcome limitations related to wave front distortions, scattering, slow acquisition, photobleaching and photodamage, which limit its spatial resolution, contrast as well as its time-resolution.
Th...

Two-photon laser-scanning microscopy (TPLSM), with its advantages for deep-tissue imaging related to infrared ultra-short pulsed excitation1, has revolutionized our view on vital processes on a single-cell level by revealing motility and interaction patterns of various cell subsets in living animals2-5. However, current technology is still insufficient to elucidate the mechanisms of organ function and dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. Current technology enables only a spatial window of few hundred microns due to scattering and wave front distortion effects on spatial resolution and signal-to-noise ratio6, which are particularly obvious in the highly compact tissue of adult animals. These scattering and wave front distortion effects are due to a highly heterogeneous and anisotropic distribution of the refractive index in tissue, leading in a first step to a depth dependent deterioration of 3D spatial resolution and finally to the total loss of signal originating from ballistic excitation photons, i.e. loss of signal-to-noise ratio. In terms of bioscientific and biomedical deep-tissue applications, this means that the current technology is unable to unequivocally reveal cellular communication because poor resolution would lead to falsely positive interactions whereas the decrease of signal-to-noise ratio would cause the system to overlook some interactions between dim structures.
In order to unequivocally detect cellular interactions in a dynamic way, a highly improved spatial resolution is needed deep within the tissue. The currently introduced powerful nanoscopy techniques based on special numerical algorithms, e.g. structure-illumination approaches, on depletion of the first excited state, e.g. STED, RESOLFT, or on molecule localization, e.g. dSTORM, PALM, have found many applications in fixed cells as well as in live cell cultures7. However, in order to extend these applications to tissue sections, living tissue and organisms we still need to overcome severe technical difficulties. Two-photon excitation STED with different wavelengths as well as with a single wavelength (sw2PE-STED) for excitation and stimulated emission has been applied to improve lateral resolution in brain slices8 or in artificial matrices with embedded cells9, respectively, at the same axial resolution as standard TPLSM. Using one-photon STED, the dynamics of dendritic spines could be imaged at the surface of the brain cortex (up to 10-15 &#181;m depth) in a living Thy1 EGFP mouse at a resolution of 67 nm10. A versatile tool for developmental biology is provided by the multifocal structured-illumination microscopy, which provides two-fold improved 2D resolution. However, this technique can be used only in organisms with a low propensity of light scattering such as zebra fish embryos11. Still, none of these techniques can be applied in the highly-scattering tissue of adult animals in several hundreds of micrometers, which are crucial models for the biomedical and clinical research of diseases with onset after birth.
Independent of the approximation used to calculate the diffraction-limited wave front shape, i.e. the point spread function (PSF), after focusing through a lens, the width of the PSF along the optical axis (axial resolution) is at least three times larger than the PSF width perpendicular to the optical axis (lateral resolution)12. Wave front distortions of different orders quantified by Zernike&#39;s coefficients considerably modify the wave front shape of focused electromagnetic wave in deep-tissue imaging leading to much larger PSFs, especially along the optical axis13-15. Hence, both the diffraction laws and the wave front distortion effects point to the resolution along the optical axis as the limiting factor in deep-tissue imaging. Whereas nanoscopy techniques focus on counteracting the limits of diffraction only, a technology which improves axial resolution and contrast by counteracting both diffraction and wave-front distortion effects is needed for high-resolution intravital imaging. Ideally, this technique should be also fast enough to allow monitoring of cellular dynamics.
The real-time correction of PSF aberrations and contrast loss using adaptive optics in TPLSM has been extensively studied and improved in the past decade13,14,16-18 and it is therefore the best currently available choice leading to a better management of ballistic excitation photons14. Still, due to the fact that most wave front correction approaches used in adaptive optics are iterative and that they have to be repeated for small areas (few 10 x 10 &#181;m2) due to the high heterogeneity of the refractive index in tissue, the acquisition speed is significantly lower than necessary for imaging cell motility and communication. Moreover, the physical limit in adaptive-optics improved TPLSM is still determined by diffraction.
Spatial modulation of illumination (SPIN) and temporal modulation on the detection side (SPADE) have been theoretically proposed to be applied to laser-scanning microscopy to improve resolution. Their practical application in intravital imaging still remains to be demonstrated19.
Taken together, there is a high demand for the development of technologies, which improve the resolution for deep-tissue imaging in living adult animals. In this work, we achieve spatial modulation of the excitation pattern by controlling the scanning process in multi-beam striped-illumination multi-photon laser-scanning microscopy (MB-SI-MPLSM)20. Contrary to structured illumination approaches, in which the excitation beam cross section is spatially modulated, we use only the scanning process to achieve the spatial modulation of the excitation. By expanding the excitation to a longer wavelength, we are able to improve both spatial resolution and signal-to-noise ratio in deep-tissue of highly scattering tissue (e.g. lymph node, free-scattering path 47 &#181;m6), independently of the optical non-linear signal we detect, e.g. fluorescence, second harmonic generation or other frequency mixing phenomenon. Using this approach at excitation wavelengths up to 900 nm we are able to dynamically image cellular protein structures on the scale of few molecules in germinal centers of mouse lymph nodes. Thus, we can better visualize the interaction between antigen carrying units on the surface of follicular dendritic cells and B cells in the process of probing the antigen during the immune response in secondary lymphoid organs.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ATTO590 &#8211; NSH for coupling</td>
			<td style="width:99px;">ATTO Tec, Germany</td>
			<td style="width:119px;">AD-590-35</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">CD21/35 &#8211; Fab fragment &#8211; ATTO590</td>
			<td style="width:99px;">DRFZ, Berlin</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">The coupling reaction was performed in the central lab of our institution.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">B1-8 Jk-/- EGFP+</td>
			<td style="width:99px;">Prof. Anne Habermann, Yale Univ., CA, US</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Can be also found at Jackson Laboratories (JAX).</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">EasySep Negative Selection Mouse B Cell Enrichment&#160;</td>
			<td style="width:99px;">StemmCell Technologies, Germany</td>
			<td style="width:119px;">19854</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Polystyren beads (605), 0.2 &#181;m</td>
			<td style="width:99px;">Life Technologies, Germany</td>
			<td style="width:119px;">F8803</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">TriMScope I</td>
			<td style="width:99px;">LaVision Biotec, Bielefeld, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OPO (manual version)</td>
			<td style="width:99px;">APE, Berlin, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Ti:Sa Laser Chameleon Ultra II</td>
			<td style="width:99px;">Coherent, Duisburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">water-immersion objective lens, 20x, NA 0.95, IR, WD 2 mm</td>
			<td style="width:99px;">Olympus Germany, Hamburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
	</tbody>
</table>

highly resolved intravital striped-illumination microscopy of germinal centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

Spatial resolution in lymph nodes
The dimensions of the effective point spread function (ePSF) correspond to the spatial resolution of a microscope12. We measured this three dimensional function by acquiring the second harmonics generation signal of collagen fibers in lymph nodes by our MB-SI-TPLSM as compared to established two-photon laser scanning microscopy techniques, i.e. field detection TPLSM (by means of CCD cameras) and point detection TPLSM (by means of PMTs).
<p...

Watch this Scientific Journal Video about Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers at JoVE.com

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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JoVE Journal

Immunology and Infection

9.9K visualizações.  Leibniz Institute.  De alta resolução de imagem intravital com melhor contraste de até 120 mM profundidade em linfonodos de camundongos adultos é conseguido através espacialmente modular o padrão de excitação de um multi-focal microscópio de dois fotões. Em 100 microns de dimensão medimos resoluções de 487 nm (laterais) e 551 nm (axial), evitando, assim, os limites de espalhamento e difração. 