OSA / NP 2010 a730_1.pdf NME15.pdf
Multiphoton Microscopy for Intravital Imaging Applications Ana M. de Paula Departamento de Física-ICEx, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil
[email protected]
Jens V. Stein Theodor Kocher Institute, University of Bern, Freiestr. 1, 3012 Bern, Switzerland
Gustavo B. Menezes, Fernanda M. Coelho and Mauro M. Teixeira Departamento de Bioquímica e Imunologia-ICB, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil
Abstract: Intravital microscopy provides a unique opportunity to study biological phenomena in living organisms. We show results for multiphoton microscopy by two-photon absorption and second harmonic generation processes applied to intravital imaging of immune cells migration. ©2010 Optical Society of America OCIS codes: (180.2520) Fluorescence microscopy; (190.4180) Multiphoton processes; 180.6900 Three-dimensional microscopy; (190.4160) Multiharmonic generation
1. Introduction Multiphoton spectroscopy and microscopy has been widely used to imaging biological and nano-structured materials. More recently it has been a unique tool applied to study biological phenomena in a living organism: intravital imaging [1]. Several nonlinear phenomena are exploited to obtain the images, such as fluorescence by multiphoton excitation or second and third harmonic generation [2]. Multiphoton fluorescence imaging, by the quasi-simultaneously absorption of multiple photons presents several advantages as compared to confocal fluorescence microscopy, there is absorption only in the focus eliminating the necessity of a pinhole and reducing the photo damages and photo bleaching outside the focus. Also the use of lower energy photons (near-infrared) permits deeper penetration into scattering samples. The other non-linear phenomenon applied to biological imaging is second and third harmonic generation (SHG and THG). It explores the non-linear optical properties of specific molecules possessing the correct symmetry. Several tissue components are known to exhibit SHG which can be used to generate label free images providing images of biological tissues without the influence of external labelling molecules. We present an experimental setup for intravital multiphoton microscopy based on a modified Olympus confocal microscope using a femtosecond Ti:sapphire laser. We show results of immune cells migration in organs, such as the cremaster muscle and a lymph node. 2.Materials and Methods Mice: C57BL/6 mice were used throughout the experiments. The protocols used were in accordance with the guidelines drafted by the University of Minas Gerais Animal Care Committee. Mice were used between 6 and 12 weeks of age. Intravital microscopy was performed as previously described [3]. Briefly, mice were anaesthetised by intraperitoneal injection of a mixture of 10 mg/kg xylazine hydrochloride (MTC Pharmaceuticals, Cambridge, ON) and 200 mg/kg ketamine hydrochloride (Rogar/STB, London, ON). The right jugular vein was cannulated to provide maintenance of anaesthetic and for IV administration of antibodies. Small incisions were made to exteriorize the tissues, and all exposed tissues were moistened with saline-soaked gauze to prevent dehydration. Multiphoton microscopy: The experimental setup is based on a modified Olympus confocal microscope (FV300) in an up-write configuration, using a Ti:sapphire laser (Chameleon, Coherent) with 140 fs pulses in the wavelength range of 680 nm to 1080 nm. The Ti:sapphire beam size is optimized to match the green laser of the confocal microscope. Most of the images presented were obtained for an excitation wavelength of 840 nm. Mice were placed in an adjustable microscope stage at home-made acrylic plates. Body temperature was maintained at 37°C using an infrared heat lamp. 3. Results Figure 1 presents images of a mouse lymph node showing lymphocytes (light green) moving throw a blood vessel (in red). The green lines is from SH generation in the collagen fibres. Fig. 1 (a) to (d) shows frames at a time separation of 40 seconds. Note the crawling movement of the lymphocyte inside the vessel wall.
OSA / NP 2010 a730_1.pdf NME15.pdf
Fig.1. Intravital image of a mouse lymph node. Green lines: SH from collagen fibres, Red: blood vessels, light green: lymphocytes Figure 2 shows images of leukocytes moving in a cremaster venule. Fig. 2 (a) to (c) shows frames at a time separation of 2 minutes, and fig. 2 (d) shows an overlay of times at every minute 1 to 9, as marked by the number under the cell.
Fig.2. Cremaster muscle: Intravital image of mouse cremaster muscle. Green cells: FITC-anti-GR1 positive cells (neutrophils), Red: PE-anti-PECAM-1 positive cells (endothelial cells) 4. Discussions and conclusions We presented an intravital multiphoton microscopy setup for imaging biological phenomena in living organisms. Results showing immune cell migration in some organs, cremaster muscle and lymph node, provides unique information for inflammatory diseases studies. Detailed study of leukocytes movement in the blood vessels allows us to observe cells trafficking in real time that can be identified by lineage specific probes eg., fluorescent antibody anti-GR1 for neutrophils, anti-CD3 for T cells. Also new lineage of specific GFP mice allow for tracking of subsets of leukocytes (eg. GFP-NKT cells). In addition, these images have sufficient resolution to examine protein distribution in blood vessels, for example, an adhesion molecule for leukocytes (PECAM-1) on endothelial cell junctions. Reconstruction of blood vessels in 3-D (Z-stacks) allows us to visualize cell-cell interactions as well as where leukocytes traverse blood vessels. Finally we are able to visualize cells outside the blood vessels, which was not possible before the implementation of this methodology. 5. References [1] Michael J. Hickey and Paul Kubes, “Intravascular immunity: the host pathogen encounter in blood vessels,” Nature Reviews Immunology 9, 364-375 (2009). [2] W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proceedings of the National Academy of Sciences 100, 7075–7080 (2003). [3] G. B. Menezes, W. Lee, H. Zhou, C. C. M. Waterhouse, D. C. Cara and P. Kubes, “Selective down-regulation of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10,”. J. Immunol, Published online Nov 16, (2009) doi:10.4049/jimmunol.0901786 Acknowledgements: We acknowledge financial support from Fapemig and CNPq
