RESUMO
In this Letter we propose a fast off-axis hologram autofocusing (AF) approach that is based on the redundant data elimination by the critical resampling of the contained complex field. Implementation of the proposed methodology enables the real-time AF with up to 12× speed-up factors in comparison to the classical approach. The method is further extended for single-shot physical autofocus of the fluorescence imaging channel of multimodal imaging instruments capable of off-axis hologram acquisition.
Assuntos
Algoritmos , Holografia/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Processamento de Sinais Assistido por Computador , Reprodutibilidade dos Testes , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.