The overall goal of this procedure is to obtain high definition infrared images of tissue samples. This is accomplished by first sectioning tissue samples onto infrared compatible slides. The second step is to set up a high definition imaging apparatus by installing the appropriate objectives.
Next, the background of the substrate is collected and the tissue sample is scanned. The final step is to use appropriate software for data processing and visualization. Ultimately, high definition infrared imaging is used to visualize and obtain biochemical information from biological tissues in a non perturbing fashion.
The main advantage of this technique over existing methods like light microscopy, is that the inherent biochemistry of the tissue can be studied without the use of dyes or probes. This method can help answer key questions to the field pathology, such as predicting recurrence of diabetic nephropathy and classifying the progression of liver disease through HEPA car osteogenesis. The implications of this technique extend towards disease diagnosis and prognosis, given that it provides vast amounts of biochemical information not available from traditional histopathology.
Though this method can be used for diagnosis. It can also be used to track the changes in the process of wound healing and identify key tissue features such as stem cells in the GI tract and the brain. First section of formal and fixed paraffin embedded tissue block at four micrometer thickness onto an IR compatible slide using a microtome.
Following this. Purge the FTIR microscope and spectrometer using dry air to remove atmospheric water from the system. Then cool both the focal plane array detector and the internal mercury cadmium Telluride detector in the microscope using liquid nitrogen mount the sample slide on the microscope stage for FTIR imaging after ensuring that the visible light is on, focus on the sample using the sample capture program.
Next, open the bundle software package and click collect. Click diagnostics and select a line spectrometer. Then click on imaging setup to calibrate the system.
In the optics tab, select detector as ground microscope detector as left, and then select transmittance under optics mode. Click setup, which will open up the lancer control window for transmission mode in lancer control. Click on raw using the stage control joystick and watching the live view of the FTIR Interferogram image move to a clean area of the slide.
At this point, adjust the integration time to approximately 8, 000 counts and the bottom condenser objective. To increase the number of counts to its maximum watch the shape of the bottom right image in lancer control to ensure that it is Gaussian in appearance and relatively uniform. After adjusting the integration time, again, move the stage to find a piece of tissue with structure, preferably the edge of a tissue.
Then perfect the focus of the image. Using the stage control joystick, move to a clean area of the slide. Press the calibrate button after selecting.
Okay, twice In the optics tab, select detector equals MCT and microscope detector equals right. Then click setup. Once the FTIR interferogram is visualized on the screen, click on find center burst and okay.
In the optics tab, reselect detector equals ground microscope detector equals left. Then select setup. After ensuring the image is still on a clean area in lancer control, click calibrate again and okay.
To collect a background FTIR image, go to the electronics tab and select an appropriate spectral resolution, which is typically of four or eight inverse centimeters. For tissue, go to the background tab and type 128 in scans to coad. Select the new file button and place the background file in the appropriate folder.
After clicking background and waiting for the scan to finish, confirm where to save the file. Click a region on the background, FTIR image and check the spectrum. At this point, click setup, and in lancer control, use the live IR view.
To find the area of interest, go to the electronics tab and type the number of scans to coad. Then click scan To prepare the FTIR microscope for high definition analysis, screw in the high magnification objective in place of the 15 x objective. At this point, open image processing and analyzing software and load the IR data file.
Apply a baseline correction algorithm to the IR data by selecting spectral tools, and scroll down and click absorbent spectra. When the pop-up menu appears, select baseline correction. Perform spectral normalization by selecting normalized spectra under the absorbent spectra menu options.
Following this, observe a list of all the IR frequencies collected within the image. Click on the frequencies that correspond to specific biomolecules to observe an image of the tissue at the selected frequency to create images that will allow visualization of different biomolecular components. Click on spectral tools and then select peak height ratios.
Scan the corresponding adjacent stained tissue section using a separate whole slide imager system that captures brightfield images alongside the IR image. Bring up the digital image of the stained tissue with the visible image program. Next, right click on the image at a region of interest and select Z profile to give the spectral information at the selected pixel.
To mark specific pixels on the image, right click on the image and select ROI tool. Create the classes that are to be labeled, for example, Meum and Bowman's capsule classes. Then select ROI type point following this, select the class to select pixels for and draw on the appropriate pixels on the IR image.
Derive the average spectra for each of the classes using the average ROI tool. Finally, compare derived spectra by plotting. In graphing software, FT IR imaging allows for the derivation of IR images of tissue that can give different contrasts depending on IR frequency.
Every pixel is composed of the entire spectrum with different peaks corresponding to different biomolecules that can give information about the biochemical properties of cell types or disease states. FTIR instrumentation has evolved from measuring in a single point mapping mode using opaque apertures to imaging mode using CASA grain objectives using either an illuminating objective, coupled with a collecting objective in transmission mode, or a single objective that both illuminates and collects in reflection mode. The advances in spatial resolution for tissue imaging have been of critical importance as cell types and tissue structures can now be identified.
In this case, the kidney glomeruli functional units were observed in a liver tissue core. It is possible to visualize hepatocytes and regions of infiltrating fibrosis that divides two distinct areas of dysplasia and non dysplastic cirrhosis. Increased spatial resolution allows isolation of specific structural features that may be chemically modified by disease before histological changes are apparent.
Biochemical changes in kidney glomerular structures such as the Bowman's capsule, meum, glomerular basement membrane, and tubular basement membrane can be identified through FTIR imaging. While attempting this procedure, it's important to remember to fully de deperate slides before scanning Following this procedure. Other methods like traditional immunochemical analysis can be performed on the same tissue section in order to correlate the biochemical signatures and tissue morphology.
Our first development, this technique paved the way for researchers in the field of tissue imaging to explore the biomolecular status of small cell types and structures within tissues. After watching this video, you should have a basic understanding how to obtain high definition FTR images of tissue samples, and perform basic spectral analysis. Don't forget that working with liquid nitrogen can be extremely hazardous and safety precautions, such as cryo safe gloves and safety goggles should always be taken while performing this procedure.
