Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

The overall goal of this method is detection of protein aggregate deposits in both clinical and scientific setups. Hepta formyl thiophene acetic acid staining and analysis of tissue from animal models of prion disease and Alzheimer’s disease is described. This method can help answer key questions in the amyloid field, such as the presence of small amounts of protein aggregates and their intrinsic conformational variations.

The advantage of this technique is that it is more selective and more sensitive than other conventional techniques. The implications of this technique extend toward therapy or diagnosis of various protein misfolding diseases because of the possibility of tailoring the probes for desired visualization technique. Generally, individuals new to this method struggle because hFTAA requires such low staining concentration.

HFTAA also displays a longer excitation and emission wavelength compared to conventional dyes. Prepare the luminescent conjugated oligothiophene solution by resuspending lyophilized hFTAA in two millimolars sodium hydroxide to prepare a one milligram per milliliter stock solution. Transfer the stock solution to a glass vial and store at four degrees Celsius.

If formalin-fixed, paraffin-embedded sections are used, deparaffinize in xylene overnight. On the day of staining, immerse the sections in consecutive baths of 99%ethanol, 70%ethanol, dH2O, and PBS for ten minutes each time. Then, allow the tissue sections to dry under ambient conditions.

While the tissue dries, prepare a working solution of hFTAA by diluting the stock one to 10, 000 in PBS. When the tissue is dry, add droplets of the hFTAA working solution to each tissue section to cover it. Incubate for 30 minutes at room temperature.

Rinse off the staining solution with 500 microliters of PBS and then immerse the slide in the PBS bath for 10 minutes. After allowing the section to dry under ambient conditions, mount using fluorescence mounting medium. Allow the mounting medium to settle overnight.

Amyloid detection can be performed directly after mounting, or even without mounting. However, if the goal of the experiment is to collect high-quality spectral information, overnight incubation is preferred. Open the microscope software, name the project, select spectral image, and start acquisition.

Select the object of interest through the ocular using the 436 nanometer excitation filter and shift the light path to the camera. In the case data manager, select the sample type spectral, label the sample, and press acquire. The acquisition window will open.

Under spectral imaging, open the settings menu, select acquisition properties, and set the spectral range to 460 to 700, the speed quality at maximum speed, and the measurement type to gas laser narrow filters. Close the dialogue box. In the image menu, select live full.

In the icon’s bar, turn fringes off. Select a region to image and ensure that the peak memory value is below 800 megabytes. Set the exposure time to a value that gives a total image brightness between 1, 000 and 3, 000.

In the icon’s bar, press the colored camera. Acquisition will start. When the image acquisition is complete, press save in the acquire spectral image dialogue box and new cell in the case data manager dialogue box.

From the case data manager, open the collected image using start analysis button. The data analysis window will open. Spectral information can be collected from each pixel of the image by selecting ROIs using the spectral display dialogue box.

Choose define and select ROIs from the relevant areas of the image. Save the spectral data as a text file using the Lib button. The saved txt file can be imported to any analysis software of choice.

Open the microscope software and begin by setting up the confocal microscope. Set the laser intensity to 0.2%the pinhole to one Airy unit, the frame size to 1, 024 by 1, 024 pixels, the scan speed as seven averaging over 16 scans, and the bit depth as eight bit. To collect the emission spectrum, select lambda mode and the argon laser set to 488 nanometers.

Collect emission between 499 and 691 nanometers using 22 channels in the 32 channel gasp detector. Set the gain to 755. Click live button and change the palette to range indicator and adjust gain to allow for non-overloaded pixels in red.

Click stop button and then capture image by snap button. To achieve single channel images, select the smart setup option. Select FITC and Alexa 532.

Select linear unmixing and apply. Adjust gain to allow for non-overloaded pixels. For FITC, set the gain to 693, and for Alexa 532, set the gain to 433 during live mode.

Click stop button and then capture image by snap button. Switch the microscope to FLIM mode. Set the pinhole to 20, the excitation wavelength to 490 nanometers, and the laser intensity to 0.5%Use pulsed lasers at 40 megahertz.

In the FLIM software, set up photon counting over 550 nanometers. In the display parameters window, follow the photon counting until the max count is around 4, 000 photon counts. Save the file and export it as SPC image.

This image shows Mallory-Denk bodies consisting of keratin aggregates in liver hepatocytes counterstained with DAPI. Here, P62 positive inclusions are shown in sporadic inclusion body myositis skeletal muscle tissue. This image shows an amyloid inclusion of islet amyloid polypeptide in human pancreas.

An amyloid deposit of immunoglobulin light chain in human intestine is shown here. This image shows deposits of prion protein aggregates of sheep scrapie in mouse brain. Prion protein aggregates in a mouse brain infected with chronic wasting disease are shown here.

This image shows A-beta-amyloid plaques in APP23 mouse brain. And this image shows A-beta pathology in APP-PS1 mouse brain. These fat biopsy smears from diagnostic samples of transthyretin amyloid in human patients graded one to four according to standard Congo Red scoring.

The yellow areas show hFTAA stained transthyretin amyloid deposits, and blue is autofluorescence from adipose tissue. While attempting this procedure, it’s important to remember to stain at adequately low concentration. Also remember to use long-pass filters to get the maximum contrast and also avoid autofluorescence and background staining fluorescence.

Following this procedure, other methods like immunofluorescence can be performed in order to identify the aggregated protein and to identify coaggregated proteins. After its development, this technique paved the way for researchers in the field of amyloidosis to explore protein aggregate structures and for organic chemistry to design novel probes for these targets. After watching this video, you should have a good understanding how to do the LCO staining to imaging each with different techniques.