Optical sectioning in differential interference contrast microscopy

Abstract

While developing a three dimensional measurement technique by the retardation-modulated differential interference contrast (RM-DIC) microscope, we found a problem. The problem is that the measurement range is restrict in λ because it applies weak phase approximation. To overcome this drawback, we propose a three dimensional (3-D) reconstruction method with z-axis scanning. This technique needs high optical sectioning like confocal microscopy. We investigate the characteristic of optical sectioning in DIC microscope, then, we confirm that DIC microscope has high optical sectioning, experimentally. We also confirm that RM-DIC microscope has higher one. Combining the optical sectioning of RM-DIC microscope and z-scanning, we develop a new 3-D reconstruction method. This novel technique overcomes the problem, the measurement range goes micron order.

Quantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation

“…The computation subtraction of images with different biases is employed in various techniques, such as polarization modulation DIC (PM-DIC) [7,8], differential detection DIC (D-DIC) with polarizing beamsplitter [9], phase-shifting DIC (PS-DIC) [10–12], retardation modulation DIC (RM-DIC) [13–15], and orientation-independent DIC (OI-DIC) [16–18]. In particularly, the PM-DIC removes a background contribution that is insensitive to defocus [8].…”

“…In particularly, the PM-DIC removes a background contribution that is insensitive to defocus [8]. It is shown theoretically and confirmed experimentally that a RM-DIC microscope has stronger optical sectioning than a conventional DIC microscope, and the optical section depth is thinner if a Nomarski prism with smaller shear amount is used [15]. Our experiments with the OI-DIC microscope using 100×/1.3NA oil immersion objective lens demonstrated the optical section depth about 0.1 μm.…”

Differential Interference Contrast Microscopy (DIC)

“…In particular, the PM-DIC removes a background contribution that is insensitive to defocus [12]. It is shown theoretically and confirmed experimentally that an RM-DIC microscope has stronger optical sectioning than a conventional DIC microscope, and the optical section depth is thinner if a Nomarski prism with smaller shear amount is used [19]. Our experiments with the OI-DIC microscope using 100 3 /1.3NA oil immersion objective lens demonstrated the optical section depth about 0.1 μm.…”

Using liquid crystal variable retarders for fast modulation of bias and shear direction in quantitative differential interference contrast (DIC) microscope

“…In particularly, the PM-DIC removes a background contribution that is insensitive to defocus 3 . It is shown theoretically and confirmed experimentally that a RM-DIC microscope has stronger optical sectioning than a conventional DIC microscope, and the optical section depth is thinner if a Nomarski prism with smaller shear amount is used 10 . Our experiments with the OI-DIC microscope using 100x/1.3NA oil immersion objective lens demonstrated the optical section depth about 0.1 μm.…”

“…The computation subtraction of images with different biases is employed in various techniques, such as polarization modulation DIC (PM-DIC) 2,3 , differential detection DIC (D-DIC) with polarizing beamsplitter 4 , phase-shifting DIC (PS-DIC) 5-7 , retardation modulation DIC (RM-DIC) 8-10 , and orientation-independent DIC (OI-DIC) 12,13 . In particularly, the PM-DIC removes a background contribution that is insensitive to defocus 3 .…”