Investigating the relationship of collagen in tonsillar disease

Session

Poster Session

Authors

Kay Polland (3), Tash Kunanandam (2), Catalina Florea (2), Catriona Douglas (1), Gail McConnell (4)

Affiliations

1. Department of Otolaryngology – Head and Neck Surgery, Queen Elizabeth University Hospital, Glasgow, UK
2. Department of Otolaryngology – Head and Neck Surgery, Royal Hospital for Children, Glasgow, UK
3. Department of Physics, SUPA, The University of Strathclyde, Glasgow, UK
4. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK

Keywords

Second harmonic generation, microscopy, multiphoton, collagen, palatine tonsils, tonsil, obstructive sleep apnoea, acute recurrent tonsillitis

Abstract text

Our hypothesis is that fibrosis, which is an excess of collagen in tissue, may play a role in tonsillar diseases including acute recurrent tonsillitis (ART) and obstructive sleep apnoea (OSA). We have used label-free second harmonic generation microscopy to assess the spatial location and quantify the relative area fraction of the mature and immature collagen in tonsil tissue from patients with ART or OSA.

Second harmonic generation (SHG) was first demonstrated in the 1960’s using a pulsed ruby laser that was projected through a quartz crystal. [1] SHG is a non-linear optical process in which two incident photons of a single frequency are destroyed and a single photon is produced with double the initial frequency. [2] Since then, SHG imaging has been used as a tool for assessing fibrosis within a multitude of organs within the body but has not been applied thus far to the study of palatine tonsils for OSA or ART. By generating forward and backwards SHG signal the mature and immature collagen within the palatine tonsil can be imaged respectively. Fibrosis can affect the function of organs within the body drastically and have a significant impact on overall health and quality of life. [3] The aim of this study is to use SHG microscopy to identify the spatial location and quantitatively assess the amount of mature and immature collagen within tonsils from paediatric patients with ART or OSA to further our understanding of the relationship between collagen types and these diseases. This could lead to a better understanding of appropriate treatment methods and may present opportunities for a new diagnostic tool to distinguish between diseases.

Samples were collected from the Royal Hospital for Children, Glasgow, UK after routine tonsillectomy. They were frozen in CellPath optimal cutting temperature (OCT) embedding matrix and stored at -80°C, sliced to 20 µm thick and mounted on slides. The slices were fixed in 4% paraformaldehyde for 20 minutes followed by 3 washes of phosphate buffered saline for 5 minutes each. Coverslips were then mounted using vectamount. For seven patients 1 tonsil slice with 5 regions of interest were imaged. Images were acquired using a Leica SP5 equipped with a 20x/0.75 numerical aperture plan achromat objective. A femtosecond-pulsed Coherent Chameleon Ti:Sapphire laser source was set to a wavelength of 850 nm and used for second harmonic generation from the collagen and two-photon excitation of autofluorescence from the tonsil tissue. Forward and backwards SHG signals, corresponding to immature and mature collagen respectively, were each detected between 420 – 430 nm, and autofluorescence of the tissue was detected between 505 – 751nm. Stitching and tiling of tissue sections was performed using the Leica SP5 microscope to increase the imaged area.

Images of tissue autofluorescence, immature collagen, mature collagen and a composite image can be seen below in figure 1.

Figure 1 a) tissue autofluorescence, b) immature collagen, c) mature collagen and d) a composite image of (a), (b) and (c). These images have been contrasted using CLAHE within Fiji [4] for presentation purposes. 

Image analysis of raw data was performed in Fiji by thresholding the images and measuring the area fraction of the image, and measuring the relative abundance of immature and collagen in each specimen. [4] We are yet to unblind the data to the patient disease (this will be done in March 2024), but we have observed two distinct populations in the analysis: one group has significantly more immature than mature collagen, while the opposite is true for the second group. We will report at the conference whether this difference is indicative of disease type.

We have used a SHG microscopy to reveal, for the first time, the location and relative abundance of mature and immature collagen in palatine tonsils from patients with ART or OSA. Our analysis shows a potential relationship between the disease types and the amount of mature collagen compared to immature collagen. Imaging of additional specimens is currently being performed to increase the sample size: once this is complete, we will unblind to disease type and reveal whether there is a statistically significant difference between the amount of collagen and the two diseases.

References

[1] Franken, P. A., Hill, A. E., Peters, C. W., & Weinreich, G. (1961). Generation of optical harmonics. Phys. Rev. Lett., 7, 118

[2] Paschotta, R. (2023). Frequency Doubling. RP Photonics Encyclopedia. Retrieved 2023-12-18 https://doi.org/10.61835/7jb

[3] De Vries, J., Kessels, B. L. J., & Drent, M. (2001). Quality of life of idiopathic pulmonary fibrosis patients. European Respiratory Journal, 17(5), 954-961. 

[4] Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682