雙光子螢光顯微技術觀察生物組織內熱傳播現象

Abstract

熱在生醫領域的應用受到越來越廣泛的重視。舉例而言：對病患受傷部位附近的局部組織加熱以加速新陳代謝，可使傷口恢復的方式；或是透過小區域以高溫加熱方式殺死體內病變組織或癌細胞，替代傳統放射性治療的療程；此外，相較於透過高溫殺死細胞，冷凍手術則是以極低的溫度讓欲切除的細胞組織壞死，這樣的好處是壞死脫落處的傷口較小，可以減少出血情形以及降低病人的疼痛。以上種種不論是利用高溫或低溫，都是屬於熱療法的範疇；而它們的機制均是針對患者身體一小部份的組織，造成不均勻的溫度梯度分佈，以達到醫師或治療師所希望達成的某些目的。針對熱療法的最佳化以及減少非必要的組織傷害，需要比較好的溫度影像。最直接獲得熱影像的方式便是插入熱電偶來獲得接觸部位的溫度，但這種方法具有侵入性，而且空間上的解析度受限於探針尖端的尺寸。紅外線熱影像是另一種發展許久的技術，亦有其限制：除了因為繞射極限造成空間解析度受限之外，只能得到表面的溫度影像資訊是最大的致命傷。 承上，我們提出了問題：「是否可以找到某種溫度相關的物理量變化機制，可以在呈現熱影像時，具備高空間解析度，以及非侵入式探測的性質？」 由於螢光分子受到熱影響會改變其發光強度，換句話說螢光強度對溫度有相依性；而另一方面，在生物組織內有許多分子具有自發螢光的特性，例如：膠原蛋白、NAD(P)H、黑色素、葉綠素…等等。結合螢光本身對溫度有關聯的特性，以及生物組織內含有數種自發螢光分子這兩件事實，暗示了只要釐清螢光強度和溫度之間的關係，我們將可以做到利用光學方法觀察熱在生物組織內的流動。由於葉綠素分子的激發波長最符合本實驗室已經具備的光源，同時葉子也是相對容易取得的樣品，因此選用葉子作為本實驗的樣本。 過去，有研究指出，葉子所產生的自發螢光強度會隨著溫度的改變而變化，藉由量測自發螢光的強度，可以推論溫度的高低。啟發自此研究，本篇論文的主要目的為利用雙光子螢光顯微術，以光學非侵入式的方法，於葉片內取得具有細胞解析度等級的三維立體螢光強度影像。並藉由建立螢光強度與溫度轉換模型，將螢光強度的空間分佈影像，轉換為溫度梯度分佈影像。透過此溫度梯度的變化分析，可以進一步提供估計組織內熱致相變化的能力。我們的方法提供的空間解析度為1微米，溫度解析度為0.6oC的非侵入式三維立體熱影像，是當前臨床所採用的熱影像方法所無法企及的。激發螢光的方式採用雙光子螢光激發，最主要的原因在於這樣非線性的激發方式提供了細胞等級的三維成像可能，這是目前的熱影像方法所無法企及的。另外透過螢光變化，還提供估計熱致相變化的能力。總結以上，本篇論文主要的方向在於建立一套模型，藉由分析葉子螢光強度的改變，可以觀察熱在組織內的傳播狀況，尺度上可以達到優於細胞等級的空間解析度，0.6oC的溫度解析度，並且具備三維的成像能力。這個方法可以提供非侵入式以及高解析度的探測，對於釐清和熱相關的生物物理機制將會有許多助益。

The applications of heat play more and more important roles in biomedical field. For example, applying heat to injured parts will be able to speed up the metabolism, and accelerate recovery. High temperature can kill malignant cell instead of radiation treatment. Cryosurgical procedure is a technique which employs the use of cooling to destroy cells, with the advantage of less bleeding and reducing pain. All of the above belong to field of thermal therapies. To optimize thermal therapy and reduce thermal damage, we need better thermal imaging. A straightforward way to measure the temperature is thermal couple which is invasive and is limited to single-point detection. Infrared thermography is a convenient way to observe thermal imaging but limited to surface image. Hence, we would like to know that “ Are there temperature-related mechanism to present thermal imaging with the capability of high spatial resolution and non-invasiveness ? ” There exist some correlation between fluorescence intensity and temperature. In addition, there are many molecules inside bio-tissue emitting auto-fluorescence, such as collagen, NAD(P)H, melanin, chlorophyll. Following these two facts, it thus comes to us that when we know the relationship between fluorescence intensity and temperature inside bio-tissue, we are able to observe heat propagation in bio-tissues by optical method. Our lab have already equipped with the light source matched the excitation wavelength of chlorophyll and also leaves are much easier to acquire, hence we finally choose leaf as our experimental sample. According to literature, it is known that auto-fluorescence intensity of leaf is strongly dependent on local temperature, we use leaf as our sample to visualize thermal imaging. In this study, we demonstrate by using time-lapsed two-photon fluorescence microscopy (2PFM), heat flow in biological tissues can be monitored based on temperature-induced fluorescence change of endogenous molecules. Compared with conventional infrared thermography or other thermal imaging tools, 2PFM provides both sub-micrometer spatial resolution and optical sectioning capability inside tissues. From the analysis of temporal variation in fluorescence, not only detailed temperature distribution can be quantified, but also the energy absorption in bio-tissue could be estimated. In this research, we have achieved the prospect of observing heat propagation in bio-tissues with spatial resolution better than cellular scale, 0.6oC thermal resolution and 3-dimensinal capability! Such a technique will be useful for the characterization of heat-related biophysical mechanisms with high spatial resolution in thick tissues non-invasively.