Parameter-optimized digital holographic microscope for high-resolution living-cell analysis.
A parameter-optimized off-axis setup for digital holographic microscopy is presented for simultaneous, high-resolution, full-field quantitative amplitude and quantitative phase-contrast microscopy and the detection of changes in optical path length in transparent objects, such as undyed living cells. Numerical reconstruction with the described nondiffractive reconstruction method, which suppresses the zero order and the twin image, requires a mathematical model of the phase-difference distribution between the object wave and the reference wave in the hologram plane. Therefore an automated algorithm is explained that determines the parameters of the mathematical model by carrying out the discrete Fresnel transform. Furthermore the relationship between the axial position of the object and the reconstruction distance, which is required for optimization of the lateral resolution of the holographic images, is derived. The lateral and the axial resolutions of the system are discussed and quantified by application to technical objects and to living cells.
Carl
Daniel
D
Laboratory of Biophysics, University of Münster, Robert-Koch-Strasse 45, D-48129 Münster, Germany. dcarl@uni-muenster.de
Kemper
Björn
B
Wernicke
Günther
G
von Bally
Gert
G
D000465:Algorithms; D016000:Cluster Analysis; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006696:Holography / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D008113:Liver Neoplasms / Q000473:pathology*; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D010363:Pattern Recognition, Automated / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D014407:Tumor Cells, Cultured