Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Summary

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

Abstract

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 µm 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 – on the level of a few protein molecules in germinal centers.

Introduction

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 animals^2-5. 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 o....

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Protocol

Multi-beam Setup for Striped-illumination TPLSM

The setup used here is a specialized multi-beam two-photon laser-scanning microscope, as described previously^6,20. 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 be a lower acquisition speed, however similar to the acquisition speed in standard PMT-based TPLSM. In order to achieve optimal quality so far as res....

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Results

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).

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Discussion

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.

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Materials

Name	Company	Catalog Number	Comments
ATTO590 – NSH for coupling	ATTO Tec, Germany	AD-590-35
CD21/35 – Fab fragment – ATTO590	DRFZ, Berlin		The coupling reaction was performed in the central lab of our institution.
B1-8 Jk-/- EGFP+	Prof. Anne Habermann, Yale Univ., CA, US		Can be also found at Jackson Laboratories (JAX).
EasySep Negative Selection Mouse B Cell Enrichment	StemmCell Technologies, Germany	19854
Polystyren beads (605), 0.2 µm	Life Technologies, Germany	F8803
Equipment
TriMScope I	LaVision Biotec, Bielefeld, Germany
OPO (manual version)	APE, Berlin, Germany
Ti:Sa Laser Chameleon Ultra II	Coherent, Duisburg, Germany
Water-immersion objective lens, 20X, NA 0.95, IR, WD 2 mm	Olympus Germany, Hamburg, Germany