顯微鏡用螢光濾光片

成像資源指南第9.3部份

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螢光顯微鏡 是利用螢光進行鏡檢的光學顯微技術，即，它使用螢光源來誘導，而不是吸收、散射或反射。螢光源是一種螢光染料，用於透過使用螢光標籤來標記蛋白質、組織和細胞，以便使用螢光顯微鏡進行檢查。螢光源的工作原理是，吸收某個特定波長區域的能量，又稱為 激發範圍，並在另一個特定波長區域內再發射出該能量，通常被稱為發射範圍。

螢光顯微鏡系統可以非常簡單，例如反射螢光顯微鏡；也可極其複雜，例如共焦系統與多光子影像系統。無論是簡單還是複雜，螢光顯微鏡採用的基本概念都是一樣的，
即：激發能量的作用是照射樣本，使其產生發射能量，雖然較弱，但卻是可定量的光波能量。激發波長和發射波長分別採用不同的中心波長，並允許使用專門的光學濾光片，從而增加整體對比度和訊號。

圖 1: 基本螢光顯微鏡架構。

圖 1所示為最基本的概念和示意圖。濾光片配置由三個特定濾光片形成，即：激發濾光片、二向色濾光片和發射濾光片。

Filter #1: 激發濾光片

激發濾光片被放置於螢光顯微鏡的照明路徑中，用於濾除光源中的所有波長。 The filter minimum transmission dictates the brightness and brilliance of images. A minimum of 40% transmission for any excitation filter is recommended such that the transmission is ideally >85%. The bandwidth of the excitation filter should be entirely within the fluorophore excitation range such that the center wavelength (CWL) of the filter is as close as possible to the peak excitation wavelength of the fluorophore. The excitation filter optical density (OD) dictates the background image darkness; OD is a measure of how well a filter blocks the wavelengths outside of transmission range or bandwidth. A minimum OD of 3.0 is recommended but an OD of 6.0 or greater is ideal.

Filter #2: 二向色濾光片 or Beamsplitter

T二向色濾光片以呈45°度的方向放置於激發濾光片和發射濾光片的中間，將激發訊號發射到接受檢查之螢光源，同時將發射訊號傳輸到檢測器。 Ideal dichroic filters and beamsplitters have sharp transitions between maximum reflection and maximum transmission, with a >95% reflection for the bandwidth of the excitation filter and a transmission of >90% for the bandwidth of the emission filter. Select the filter with the intersection wavelength (λ) of the fluorophore in mind, to minimize stray-light and a maximize the fluorescent image signal-to-noise ratio.

Filter #3: 發射濾光片

發射濾光片被放置於螢光顯微鏡的成像光路中，用於隔絕接受檢查之螢光源的整個激發範圍，同時傳輸螢光源的發射範圍。The same recommendations for excitation filters hold true for emission filters: minimum transmission, bandwidth, OD, and CWL. An emission filter with the ideal CWL, minimum transmission, and OD combination provides the brightest possible images, with the deepest possible blocking, and ensures the detection of the faintest emission signals. 

圖 2: 典型的激發和發射分佈。

圖 2 所示為典型的激發和發射分佈。此圖清楚地顯示了吸收和發射分佈均採用了相同的波長，這也是為什麼優質濾光片需要高穿透率、窄帶寬、高光密度和極度陡峭的止透和起透帶的主要原因之一。使用品質較差的濾光片最終會造成待測物、樣品或昂貴的傳感器損壞。聯絡我們以協助您選擇適合您應用的濾光片。

How Does EO Match Filter Pairs?

Edmund Optics offers dozens of in-stock and ready to ship optical filters that fit within the excitation and emission regions of each specified fluorophore. We make it easy to quickly search filters by fluorophore. Simply browse the recommended filters that match the peak excitation or emission wavelength, with maximum transmission at that wavelength. For fluorescence microscopy applications using multiple fluorophores, laser sources, alternate dichroic filters or beamsplitters, or applications more complex than typical fluorescence microscope setups, contact us to discuss your specifications.

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