However, the attainable space-bandwidth product (SBP) of these QPI techniques is fundamentally limited by the optical system used, resulting in a tradeoff between image resolution and field-of-view (FOV). Furthermore, due to the limited raw images (generally no more than 4 images) required for phase reconstruction, high-speed or even single-shot QPI has been demonstrated by employing fast switchable devices or using spatial/color multiplexing techniques 9, 10, 11. Various QPI techniques have been developed during the last decades, such as digital holography (DH) 6, 7, transport-of-intensity equation (TIE) 3, and differential phase contrast (DPC) 8 based methods, providing invaluable optical tools for biomedical research thanks to their unique capabilities to image optical thickness variation of living cells and tissues without the need for specific staining or exogenous contrast agents. Compared to fluorescence imaging, label-free quantitative phase imaging (QPI) approaches 3, 4, 5 are particularly attractive due to their noninvasive and nontoxic properties. High-throughput microscopy allows to high-content quantitative analysis of multiple events in a large population of cells, which is of crucial importance for many applications, such as personalized genomics, cancer diagnostics, and drug development 1, 2. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm 2, corresponding to a space–bandwidth–time product of 411 megapixels per second. The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture ( NA) in the frequency space. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |