Articular Cartilage Optical Clearing Research By Raman Spectroscopy

Articular cartilage plays an important role in joint activity,and cartilage degeneration can lead to osteoarthritis?OA?.In recent years,many studies have been achieved on the pathogenesis and pathological changes of OA.However,the hypothesis of subchondral bone-induced OA and the early diagnosis of OA have not yet made any breakthrough.Pathological Tissue assessment is the gold standard for determining whether or not patients have osteoarthritis and the severity of OA,but the pathology is invasive.Nowadays imaging methods are commonly used to provide unique and complementary information for clinical use.However,these imaging methods fail to provide molecular information on the key biochemical components of cells and their stroma.Considering that it is difficult to observe the early stages of OA by conventional means,a small-sized near-infrared Raman spectrometer has been designed in this paper.The light-transparent effect of cartilage tissue is explored by Raman spectroscopy combined with tissue optical clearing technique in order to get deep micro-information,then Fisher discriminant algorithm based on Raman spectra was used to classify and identify normal and lesion cartilage for early diagnosis.Firstly,development of small-sized high-performance NIR Raman spectrometer.After the review of the theory and current situation of Raman spectroscopy,it was found that there were some problems such as astigmatism,coma aberration and curvature of field,therefore,Raman spectrometer based on co-axial collimation and focusing imaging was proposed.Co-focal-plane external optical system matching the entrance slit of dispersion system was designed,and then used to effectively collect Raman signals and remove stray light.Using transmission-type collimation and focusing imaging method was adopted in the dispersion system,the scattered light was projected onto the grating for dispersion.The home-built spectrometer system?785nm excitation?achieved high resolution(3cm^-1),high repeatability and high sensitivity in spectral detection in a detection range of500²200cm^-1.Secondly,cartilage optical clearing research based on self-made Raman spectroscopy and tissue optical clearing technique.Raman spectra of femoral cartilage samples of dogs were collected under different clearing agent concentration and immersion time after glycerol and iohexol were selected as clearing agents.The Raman integral intensity ratio of phosphate group(920-960 cm^-1)to amide I-band(1595-1700 cm^-1)were calculated to investigate the optical clearing effect under different concentrations of glycerol?40%100%,Iohexol 50³50 mg/ml?and in different immersion time?10⁶0 min?,that is,to find a suitable clearing concentration and clearing time.It was found that the two kinds of clearing agents enhanced the intensity ratio compared to the non clearing agent.In the same immersion time,60%glycerol and 150mg/ml Iohexol can obtain good result;while at different concentrations,the clearing of glycerol were the strongest in 20min,as well as iohexol after 50 min.The in situ information of articular cartilage and subchondral bone were finally obtained.Lastly,the classification and identification of cartilage samples were performed by Fisher discriminant algorithm combined with Raman spectroscopy.134 Raman spectra of cartilage samples?42 normal,44 lesions at 8 weeks and 48 lesions at 7 months?were selected.The high dimensional data were dimensionally reduced to a lower dimension by PCA.Linear discriminant functions were constructed and compared by distance.Three types of samples were distinguished and identified.The correct rate was 100%and 99.3%for initial case and cross-validation case,respectively.In summary,the developed small-sized near-infrared Raman spectrometer has the advantages of high performance,which is convenient and flexible.In the study of optical clearing of cartilage tissue,the Raman information of the subchondral bone and the optical clearing effect of the cartilage were directly detected.Fisher discriminant algorithm combined with Raman detection achieved classification and identification among the normal cartilage and two stages of degenerated cartilages.Therefore,the research in this thesis provides an effective method and a powerful tool for the deep information monitoring of clinical diseases such as joint diseases.It is an effective way of thinking beyond the conventional detection means,which could be further extended to a potential and high efficient diagnosis method in clinical,but also could be expanded for biomedical research and other scientific fields.This research will promote the research of joint diseases and the development of biomedical spectroscopy to a great extent.