Abstract
We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.
Highlights
Imaging a live cell without staining is a major challenge
We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity
We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution
Summary
Imaging a live cell without staining is a major challenge. The most widely used non-staining technique for visualizing transparent living cells is the phase contrast method. Our laboratory has developed several forms of quantitative phase microscopy (QPM) based on interferometry techniques. QPM has provided the quantitative morphological structure of a living cell in 2D [1,2]. An imaging detector, such as a CCD, is used to capture distorted interferograms from samples, and optical phase delays are calculated. Phase delays can be used to directly compute important physical cellular properties. For samples that are fairly homogeneous, it is reasonably inferred that phase delays provide direct information about cell shape. A healthy red blood cell (RBC), which can be considered to have internally uniform hemoglobin distributions, is an important example of this
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