The present work shows design, development, and testing of the multimodal optical setup for simultaneous auto-fluorescence imaging, spectroscopy and quantitative phase microscopy (SAF-QPM) from same field of view (FOV). The SAF-QPM combines the capabilities of autofluorescence, offering molecular insights into metabolic activities, with quantitative phase imaging, providing nanometres-level sensitivity to cellular morphology and refractive index distribution. The autofluorescence serves as a biomarker for abnormal cellular changes associated with cancer development. Simultaneous incorporation of quantitative phase microscopy of the same tissue section probes the refractive index-dependent changes in the cellular morphology associated with cancer development. Non-interferometric QPI technique is used to retrieve the phase information of the sample, which overcomes the limitations of instability and coherent noise associated with interferometric QPI techniques. The incorporation of simultaneous AF and QPM from the same field of view enhances the accuracy and specificity of label-free cancer diagnosis.
Quantitative phase measurements in a Transport of intensity equation(TIE) based method requires capturing images at multiple defocussed positions. Using these intensity measurements along with the phase obtained we demonstrate an optimization algorithm that improves the quantitative phase estimation recovering the fine details that may be lost due to diffraction effects, aberrations and limitations of the TIE based method. We implement a cost minimization scheme to minimize the difference between measured and theoretically expected amplitude to improve the estimate of the required object field. Application of the method shows an improved accuracy in phase estimation of simulated and real objects.
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