In order to perform normalized born optical projection tomography, the sample is first fixed and embedded in a cylinder of auger. The sample is then rotated along a vertical axis and then uniformly illuminated with an excitation wavelength, and the transmission is measured with a C, CD suitable optics. The fluorescent signal at wavelength is measured in transillumination mode.
The acquired fluorescent and absorption images are used to determine the normalized born ratio and reconstruct the distribution of fluoro fours within the sample. Hi, my name is Claudia Naone and I work at the Centers for Systems Biology at Mass General Hospital, Harvard Medical School. I'm Danielle Raki from the Institute for Biological and Medical Imaging at the Technical University of Munich and Al Center Munich.
I am Giselle Fido. I am the director of Experimental su Co here at C-M-I-C-S-B, Massachusetts General Hospital, Harvard Med School. Today we'll show you a procedure for real time ex vivo whole organ imaging using the Born normalized approach for optical projection tomography.
We use this procedure in our laboratory to study fluorescence molecular probe distribution in hollow organs. So let's get started. This video will explain the procedure for using optical projection tomography to image prepared tissues or organs and an accompanying video.
We explain how to use this system to perform time-lapse imaging on developing drosophila. The three dimensional imaging technique of optical projection tomography requires a unique combination of optics and a rotating stage. In this segment of the video, the imaging procedure will be elucidated.
To begin, the primary light source serves as a source for both illumination and absorption measurements, and to provide fluorescent excitation for fluorescence measurements, the primary light source is first filtered with a narrow band pass interference filter. Next, the light passes through a set of fixed and variable neutral density filters. These are combined with the presence of an automatic shutter to control the amount of light on the sample and to keep it low enough to prevent any photo bleaching.
Uniform sample illumination is achieved by using a beam expander and a combined glan telescope lens with a diffuser lens. Finally, optical narrow band pass interference filters are used to optically cleanse the excitation source light. The sample is immersed in a clearing solution and held in place for imaging within a glass tube.
The sample is rotated along its vertical axis by way of a high speed rotation stage with an absolute accuracy of 0.05 degrees. Three distinct manual controllers allow for the sample vertical axis to be tilted and adjusted in its orthogonal plane. The absorbent signal is acquired in trans illumination and is directly detected by a CCD camera.
The fluorescent signal is filtered through a narrow band pass interference filter, coupled with a long pass filter and is then collected with a tele centric lens. Tele lenses offer the advantage of providing unique features that make them ideal. For optical projection tomography.
The presence of an aperture stop located within the lens assembly at the focal point of the lens is key. It assures that the image will travel parallel to the optical axis. This eliminates perspective distortion and provides equal magnification across many focal planes within the tele centric depth.
The next video segment describes a technique for preparing samples. This will be followed by methodology used for imaging. The sample tissue or organ preparation for tomographic imaging relies on an ethanol based dehydration of the sample that avoids asymmetric shrinkage.
This process takes many days to complete. In this video, a mouse heart is prepared, but the protocol can be applied to any tissue or organ. We will demonstrate our fluorescence imaging method using the heart of an animal that has sustained a controlled infarction.
In other words, a loss of blood supply leading to obvious tissue necrosis in advance. The mouse has been injected with pro since six 80 inact fluorescent sensor reporting on cathepsin activity in healing. Myocardium anesthetize the mouse for perfusion and heart extrusion using an intraperitoneal injection of ketamine and xylazine Then inject 50 units of heparin intraperitoneal to prevent coagulation.
Five minutes later, perform the longitudinal laparotomy on the fully anesthetized animal to remove the blood. First, open the left renal vein. Now slowly inject 20 milliliters of saline solution into the inferior vena cava.
The saline displaces the blood after flushing out all of the blood under a dissecting microscope. Continue using standard operating methods to open the thorax and remove the heart. Now, fix the heart for eight hours in 4%PFA at four degrees Celsius.
From this point forward, the tissue preparation steps must be performed in the dark to avoid bleaching of any fluorescent contrast agents or proteins applied to the tissue. When fixation is complete, wash off the PFA with a 15 minute rinse in PBS, the cleaned up heart is then embedded in a pool of 0.8%aros. Once the aros dries, the agarose is cut away around the tissue to make a block shape.
Next, dehydrate the aros encrypted heart through a five step series of first soaking in 20%Ethanol, then progressively higher concentrations of ethanol, and finally, pure ethanol. This dehydrating procedure helps in avoiding any asymmetric shrinkage of the sample. The first four baths in diluted ethanol must be performed for an hour and the final bath in 100%Ethanol must be performed for at least 10 hours.
Once the ethanol treatments are complete, put the sample in Murray's clear solution until the sample clears, which varies from a few hours to several days. This clearing solution mimics the cellular water and index matches cell membranes, thus making the tissue transparent. Ethanol in minimal traces can be present at the end of the clearing process, giving rise to several artifacts in the reconstructions due to thermal motion of the alcohol within the clearing solution itself.
In order to avoid this problem, a second clearing cycle is recommended. This should not take more than four hours. Once completed, the sample is ready for optical projection.
Tomography the with the aid of a computer. The reconstruction of optical absorption in the absence of light scattering can be obtained using a common filtered radon back projection algorithm. This process is analogous to two dimensional x-ray computed tomography.
Place the block of aeros into the transparent imaging chamber filled with the clearing solution. Now mount the chamber on the stage, illuminate the sample with a collated beam for both excitation and transmission measurements. The excitation source choice is based on the chosen fluorescent protein or molecular imaging contrast agent.
It is convenient to align the vertical axis of the sample parallel to the column of pixels of the CCD. Rotate the sample over 360 projections using angular increments of one degree, acquire images at each angle in trans illumination at both the absorption and fluorescence wavelengths. The software running the acquisition automatically controls the shutter on the primary light source to avoid continuous illumination and reduce sample photo bleaching.
The shutter is especially important when long integration times are required to create a 3D reconstruction from the absorption data. Simply use a filtered radon back projection algorithm for 3D reconstruction from the fluorescent images. Other absorption contributions must be taken into account, which are described in the next video segment.
After completing the steps required for an absorption reconstruction, there are a few more steps to completing a fluorescent reconstruction. The reconstruction of an optical map in the absence of light scattering can be obtained using a common filtered radon back projection algorithm. The process is analogous to two dimensional x-ray computed tomography.
When reconstructing fluorescence distribution. The varying spatially dependent absorption makes incorrect the use of the inverse radon for back, projecting the fluorescence images severely affecting the obtained fluorescent protein or fluorescent molecular probe distributions and its quantification ability. Each ray at the excitation wavelength Lambda X that is emitted from an arbitrary source position Xs, is exponentially attenuated as it passes through the imaged object.
After exciting, the fluorochrome located at position X, the radiation at the emission wavelength is filtered and collected by the CCDs pixel located at position XD using the Teleric imaging lensing system. Although the emitted fluorescence does not follow the same path as the excitation ray, the intensity recorded at each pixel approximates a projection of all the fluorochromes excited along the entire focal zone corresponding to this particular excitation ray. Due to the system's high tele centricity, the imaged object is then rotated 360 degrees and multiple source detector measurements, UX at the excitation wavelength and UFL at the emission wavelength are acquired with the CCD camera.
These are then combined under the normalized born field UB defined as the ratio of the two. The reconstruction of the fluoro chrome distribution can be facilitated by writing the forward model for both excitation and emission light propagation the greens function describing the excitation. Ray propagation follows a simple beer Lambert law where moo A is the optical absorption distribution previously determined.
The light intensity UX emitted from the source at position excess and measured by the detector at position XD on the C.C, D can be written as the product of the green function with a constant B that is system dependent. The precise distribution of the excitation light intensity GX along each ray path can then be calculated. The fluorescence intensity UFL detected at the detector XD that is stimulated by the light emitted from the source Xs takes into account the contribution of all theros that have been excited along the ray path from X to xd.
The forward model of our imaging problem can then be written as ub ub, where alpha incorporates the unknown constants that are system dependent. The forward model is then discretized on the assumed mesh and an inversion is performed to extract the fluorochrome distribution, acquire both the transmission and fluorescent signal and use the conventional back projection algorithm to reconstruct the sample. Here, transmission and conventional fluorescence optical projection tomography reconstructions are shown.
A reconstruction of a single plane of the heart is shown in both absorption and fluorescence. The normalized born reconstruction is presented and it shows the difference in the dye distribution within the heart. After normalization, We have just shown you how to be the setup for optical projection tomography and how to remove artifacts due to optical absorption in the reconstructions for fluorescence tomography.
It is in fact very important to keep in mind to clear the sample in order to remove the contribution of scattered light, but at the same time, it's also important to balance the clearing process in order to keep as much as possible of the fluorescence contribution. Another thing that is very important to remember when using this approach is to calculate the bone normalized ratio that is obtained by dividing the fluorescence images by the absorption ones. So that's it.
Thanks for joining our visualized experiment and good luck with your own experiments.
