The overall goal of this procedure is to prepare a standard two photon microscope for high resolution intra vital imaging. This is accomplished by first aligning the microscope, which includes adjusting the lasers. Next, the beam multiplexer is aligned to achieve perfect beam shape and beam lit arrangement.
In case of multibeam scanning, then the raw images are acquired by scanning the sample with the striped illumination pattern. Finally, an algorithm is applied to calculate the high resolution final image. Ultimately, striped illumination microscopy is used to show details of immune system behavior within germinal centers in the lymph nodes of mice, formerly inaccessible to intra vital microscopy.
The main advantage of this technique over existing methods like structured illumination or localization microscopy, is that it can be applied in great tissue depths in live samples. It does not require any modification of the laser beam shape, thus keeping the method very simple and easy to implement to any multi photon microscope. The setup used for multi-beam two photon striped illumination microscopy is shown here to achieve optimal resolution and contrast quality with the lowest level of photo bleaching and fastest acquisition.
Consider making adjustments to the setup as outlined in the text protocol. After making the adjustments, check that the beam arrives back to the mirror that is located directly above the entry mirror at the end of the prism based pulse compressor. After the light path is set from here, the beam is reflected into the beam multiplexer.
If performing multi-beam striped illumination, multi photon microscopy with the scanner off. Align the mirrors in the beam multiplexer by placing a homogenously fluorescing sample on the stage and focus the laser beam inside the sample, but near the top surface. Close to the objectives front lens.
Set the microscope to the multi-beam mode and choose the number of beams and the polarization. For example, P to find the laser beam by imaging the fluorescent slide, set the P shutter open and run the CCD camera continuously while focusing the beam, ensuring that it's in the center position. When aligning the P beam switch to 64 beams mode and open the corresponding shutter.
Check the position of the beam. Adjusting the first through the fifth beam multiplexer mirrors, ensuring that the beam lits are equally distant. Since striped illumination algorithms are based on this assumption, replace the uniformly fluorescent sample with the heterogeneously structured one.
For instance, Conval area root sample and scan a multi-beam image. Check that the image appears sharp, uniform and straight. Replace the heterogeneous sample with the homogenous sample.
Start the XY scanner to control the scanning process. To generate the striped image excitation pattern for multi-beam scanning. Move the Y scanner mirror perpendicular to the beam lit line.
Repeat the generated pattern here indicated by number of blocks by moving the X scanner mirror here called block shift to generate a rectangular striped image. Place the heterogeneous sample back and use the scanner as the master trigger. Synchronize the camera with the scanner to automatically acquire and save a striped image to repeatedly acquire striped images.
Move the X scanner mirror to different positions by choosing enough repetition steps here, number of shifts and the appropriate step length here. Shift step between two subsequent striped images to cover the distance between two consecutive beam widths. Set the X shift to a value that is below the lateral resolution limit at the given excitation wavelength.
For example, using 10 steps of the X scanner mirror per shift and 12 or 13 shifts leads to the best spatial resolution. Results with the conval area root slide to optimize these two values until the conval area image becomes the sharpest. Use the line profile tool on the conventional CCD images and the calculated striped illumination images.
Compare the width of the line profiles, which in this example is the fluorescent signal from conval Cell walls. Acquire each raw striped image synchronized with the scanner movement and save it separately, for example, to TIFF or binary files. To generate a two dimensional matrix of the high resolution striped illumination image, open a complete set of raw striped images as matrices in the evaluation routine.
Use either the minimum maximum algorithm or a Fourier transform based algorithm as previously described in detail to perform striped illumination, multi-beam, multi photon microscopy in deep tissue. In live organisms acquire striped images in different tissue depths over time and evaluate them as previously described, to generate a high resolution 3D movie of the dynamic interaction between germinal center, B cells, and follicular dendritic cells. The dimensions of the effective point spread function correspond to the spatial resolution of a microscope.
We measured this three-dimensional function by acquiring either the second harmonics generation signal of collagen fibers or the fluorescent signal of 100 nanometer polystyrene beads emitting at 515 nanometers in mion lymph nodes. Using our multi-beam striped illumination T-P-L-S-M as compared to established two photon laser scanning microscopy techniques such as field detection T-P-L-S-M by means of CCD cameras and point detection T-P-L-S-M. From these measurements, it becomes obvious that at the surface of the specimen, both the lateral and axial resolutions correspond to the expected values predicted by the diffraction theory.
However, with increasing imaging depth, and thus with an increasing optical path of both excitation and emission radiations through medium with highly varying refractive indices, the spatial resolution deteriorates considerably independently of the setup used. Using M-B-S-I-T-P-L-S-M independently of imaging depth, we reached approximately a 20%better lateral resolution and a 220%better axial resolution. As compared to the standard T-P-L-S-M approaches.
The clonal selection of B cells during the immune response within secondary lymphoid organs of adult mice is the first step on their way to differentiate into memory B cells or plasma cells. In this process, the highly dynamic interaction between follicular dendritic cells and B cells at the level of immune complex structures within germinal centers is believed to play a central role. Only by using a highly resolving intra vital microscopy technology is it possible to dissect this cellular communication and its implications for the immune response seen here as labeled by anti CD 2135 FAB ATO five 90 on follicular dendritic cells.
M-B-S-I-T-P-L-S-M provides for the first time the possibility of inter vitally visualizing and quantifying the dimensions of the clusters of immune complex deposits in up to 120 micron depth in germinal centers of popal lymph nodes. These images show the direct comparison between the 3D fluorescence images of immune complex deposits in a germinal center as acquired by conventional PMT based and M-B-S-I-T-P-L-S-M. Moreover, the interactions of the immune complex deposits with germinal center B cells could be highly resolved and can now be investigated in combination with intra vital functional probes for proliferation, differentiation, or apoptosis.
While attempting this procedure, it is important to keep in mind that with a camera detection based technique, the acquisition speed is approximately eight times higher, while the maximum imaging depth is approximately 25%lower than those achieved with a standard photomultiplier based multi photon microscope. After watching this video, you should have a good understanding of how to set up your multifold microscope to work with the striped illumination method, including the adjustment of the microscope light path, the alignment of the beam lats, the calculation of the scanning parameters, and the ways to calculate the final high resolution image. For intravital deep tissue experiments.
