Abstract
Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.
Highlights
High-resolution fluorescence microscopy and quantitative phase imaging of the specimen provide important information about biological processes [1,2,3,4]
We developed a multi-modal microscope, by integrating chip-based Total internal reflection fluorescence (TIRF) microscopy with a Linnik-type quantitative phase imaging (QPI). This multi-modal microscope merges the individual advantages of TIRF microscopy, such as excellent optical sectioning, low photo-toxicity and high-contrast images, together with the quantitative expression of the cellular physiology and morphology provided by QPI
Waveguide chip-based TIRF microscopy possess several advantages over conventional lensbased and prism based TIRF microscopy, including that the generation of the evanescent field is independent of the collection objective lens and multimodality, enabling TIRF imaging over an extraordinarily large FOV
Summary
High-resolution fluorescence microscopy and quantitative phase imaging of the specimen provide important information about biological processes [1,2,3,4]. It is difficult to get a high contrast image of cells using bright field optical microscopy. This has lead to widespread usage of optical microscopy techniques that can generate high-contrast images. The two most common techniques to enhance the contrast of the sample are a) phase enhanced label-free optical microscopy and b) fluorescence based optical microscopy. Phase contrast (PC) and differential interference contrast (DIC) microscopy techniques are widely employed [6,7]. DHM is a single shot technique enabling high-speed imaging and is widely used for live cell imaging applications, such as cell membrane fluctuations, cell growth and cell division [9,11]
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