改建常壓質譜游離平台以提升分子影像表現

Abstract

常壓游離質譜影像 (Ambient ionization mass spectrometry imaging)這項技術在這幾十年間不斷進步，除了可以在一大氣壓環境下提供分子在生物樣品中的空間分佈優勢以外，少量的樣品前處理準備也使這類技術備受青睞。即便如此，常壓游離質譜影像也面臨諸多限制與挑戰。比如因為減少樣品前處理 (Sample pretreatment)，所產生的基質效應 (Matrix effect)造成待測分子游離效率不佳無法被測量。此外，由於無法提供高空間解析度 (Spatial resolution)品質的圖片，生物組織中細部的化學分子組成便容易被忽略。為了解決上述兩個問題，我們重新設計常壓游離質源並引用電腦模擬運算等方法來改善質譜影像品質以及質譜訊號強度。 常壓游離質譜影像技術可用來診斷生物樣品中的分子組成以及其在空間中的分佈。然而，低空間解析度一直以來都是長壓值譜影像技術的缺點，細緻的組織紋路上的化學分子可能因為低解析度而無法在質譜影像中被清楚描繪。因此，我們將常壓質譜影像平台所收集的質譜影像與透過光學顯微鏡取得之高空間解析度影像利用影像處理以及線性組合等電腦運算工程，將兩個不同來源的影像進行融合以得到一個高空間解析的質譜影像。在此實驗中我們分別使用脫附式電噴灑游離 (DESI)以及奈米脫附式電噴灑游離法 (NanoDESI)作為我們的游離源，來分別描繪小分子如脂質以及蛋白質等大分子在小鼠器官切片中的二維分佈。相鄰的組織切片則是經過蘇木精伊紅染色處理後以光學顯微鏡拍照取得高解析度的照片，再利用電腦運算軟體將兩種圖融合並預測出高空間解析質譜影像圖。 除了空間解析度的限制，基質效應或離子抑制效應 (Ion suppression effect)也是嚴重影響常壓質譜影像品質的問題之一。由於常壓游離源是直接將生物樣品表面上所有的分子全部一起直接送進質量分析器中，在輸送過程中過多的干擾物或是背景雜訊會和待測分析離子進行競爭，導致無法被質譜儀偵測或是訊號強度差。為了移除干擾物 (Interference)以及背景雜訊 (Background noise)，我們將場不對稱離子遷移率儀 (Field asymmetry ion mobility spectrometry)與質譜儀結合，藉由場不對稱離子遷移率儀可以在氣象環境中將離子分離的特性，我們可以有效提升質量分析器分析待分析物的效能。 在此實驗中，我們成功利用改建的常壓游離質譜影像游離源將生物組織切片中的分子的分布描繪出，並且在電腦運算以及場不對稱離子遷移率儀的協助下，我們得到高空間解析度的質譜影像以及提升待測分子在質譜中的訊號強度。從實驗結果來看，我們相信新的長壓游離源非常具有潛力，有機會成為不可或缺的分析技術之一。

Ambient ionization mass spectrometry imaging (MSI) techniques enable investigating biological samples in situ, and providing their chemical distribution with minimal sample pretreatment. However, due to the coarse spatial resolution of ambient ionization MSI, and dynamic range limitation. Detailed chemical information of the sample might be missing, here we report two methods to solve the problems. To improve spatial resolution of ambient ionization mass spectrometry imaging, we utilize image fusion paradigm developed by Plas .et .al to fuse both ambient ionization MSI and optical microscopy to generate predictive high spatial resolution MSI. The multimodality paradigm for imaging fusion use multivariate regression and statistic measurement to compare and integrate two distinct image techniques into one. In this study, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) was employed to image lipids and proteins of mouse tissue sections for chemical information acquisition. Whereas H&E stained adjacent sections were employed for high quality spatial information collection. Furthermore, we integrated field asymmetry ion mobility spectrometry (FAIMS) or so called differential mobility spectrometry with a high mass resolution mass spectrometry to reduce the problem of dynamic range or ion suppression problem. We are able to deplete chemical noise of isobaric compound, background interference from the biological tissues in gas phase before they enter mass analyzer by separate characteristic of FAIMS. Most important of all, dominant compounds will be filtered away and minority chemicals will remain and be focused prior mass analyzing. A pneumatically-assisted nanospray desorption electrospray ionization (pneumatically-assisted nanoDESI) ion source was developed to assist solvent extraction efficiency for depicting spatial distribution of polypeptide in tissue sections. With the FAIMS integrated ambient ionization mass spectrometry imaging system, ion signals of pneumatically assisted nanoDESI mass spectra display diverse polypeptides or proteins compare to the traditional nanoDESI. The remodel ambient ionization mass spectrometry imaging platform demonstrate its ability provide high quality mass spectrometry images and unveil hidden protein species by integrated with complementary modalities to cover its shortage. Moreover, the platform preserves the advantage of AIMS which enable it to act as another alternative analyze tools in other applications.