Chapter 11 - Differential Interference Contrast (DIC) Microscopy

Chapter 12 - Amplitude Modulation Contrast Microscopy

Differential Interference Contrast (DIC) microscopy is a nondestructive imaging modality that has been widely used by biologists to capture microscopy images of live biological specimens. However, as a qualitative technique, DIC microscopy records specimen's physical properties in an indirect way by mapping the gradient of specimen's optical path length (OPL) into the image intensity. In this paper, we propose to restore DIC microscopy images by quantitatively estimating specimen's OPL from a collection of DIC images captured from multiple shear directions. We acquire the DIC images by rotating the specimen dish on the microscope stage and design an Iterative Closest Point algorithm to register the images. The shear directions of the image dataset are automatically estimated by our coarse-to-fine grid search algorithm. We develop a direct solver on a regularized quadratic cost function to restore DIC microscopy images. The restoration from multiple shear directions decreases the ambiguity among different individual restorations. The restored DIC images are directly proportional to specimen's physical measurements, which is very amenable for microscopy image analysis such as cell segmentation.

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.

INTRODUCTIONThis protocol describes the use of electronically enhanced, differential interference contrast (DIC) imaging techniques for the study of living cells. Methods for DIC imaging of cells using an inverted microscope and for infrared (IR) imaging of brains slices using an upright microscope are presented. DIC microscopy (also known as Nomarski microscopy) is extremely useful for resolving individual cells and cellular organelles in live, unstained tissue. The data obtained are valuable in their own right, as well as for complementing fluorescence microscopy data. DIC microscopy can be applied to cell culture, brain slices, and even intact organisms (such as embryos). High-quality images of living cells in brain slices can also be obtained using a combination of DIC optics and IR video microscopy.

Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.

Amongst the techniques suggested for the determination of Giardia cyst viability, the use of the fluorogenic dyes, fluorescein diacetate (FDA) and propidium iodide (PI) is the most often recommended, even though it appears to overestimate the number of viable cysts. In the present study, the replacement of FDA with 4',6-diamidino-2-phenylindole (DAPI) allowed simultaneous direct immunofluorescence with monoclonal antibody labelled with fluorescein isothiocyanate (MAb-FITC). Under these conditions, it was possible both to quantify the cysts according to the immunofluorescence technique, and to appreciate their viability by using fluorogenic dye staining (DAPI and PI) and differential interference contrast (DIC) microscopy. This method proved to be significantly better than the counting methods normally suggested. The technique has been applied to Giardia cysts recovered from faeces and wastewater sludge.

Nondestructive Detection of Glass Vial Inner Surface Morphology with Differential Interference Contrast Microscopy

The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca(2+) and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease - Tlag) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow Trelease, in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences. Spores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination.

Extraction of phase and amplitude information in differential interference contrast (DIC) microscopy is a challenging but stimulating task. In this paper, a simple straightforward non‐iterative method to extract the absorption and phase difference images for DIC is presented. According to the method proposed in [1], phase information can be acquired straightforwardly. The feasibility of our method is confirmed by experimental results of imaging liquid paraffin drops using a commercial DIC microscope. Compared with the available methods and previous research [1–4], our approach simplifies the procedures to extract the absorption and phase difference images without weak absorption limit.

In this study, the differential interference contrast (DIC) approach originally used for image enhancement to increase the contrast between a transparent object and the background is adopted for the dimension measurement of transparent structures. With the phase difference image retrieved using the DIC technique, the phase map of the examined object can be approximated by integrating the phase difference. The need of integration accuracy is much higher for measurement than for image enhancement. In this study, a modified Fourier phase integration is proposed to reduce the effects of noise on surface profile reconstruction. The simulation results show that the proposed approach can effectively reduce the effects of noise. Experimental results are also conducted to study the feasibility of using the transmitted DIC with the proposed integration method for transparent object measurement. The results show that the height of a transparent structure measured using the DIC method is quite close to those measured using an atomic force microscope, while those measured using the white-light interference method result in a much larger measurement than all others.

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called “non-blinking” quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

A new differential interference contrast microscopy (DICM), which uses Savart prisms as a shearing plate and a phase-shifting device, is proposed. The system consists of a phase-shifting module (PSM) and a DICM module (DICMM). The PSM has two Savart prisms: the first prism separates the incident beam into two parallel beams, and the second prism recombines these two beams. The optical path difference (OPD) of the two beams, which is represented by a biased OPD, can be adjusted by rotating the angle of the normal surface of the second prism. In the DICMM, the other Savart prism is used to replace the Nomarski prism (NP) in conventional DICM. It combines with an afocal microscopic system (AMS) to produce a Savart-DICM system, which is able to perform a phase-shifting technique by changing the biased OPD to produce a phase shift of π/2 for each step. This paper describes the configuration and measurement theory of the microscope. The experimental results confirm the validity and capability of the proposed microscope.

BackgroundThe purpose of this study was to compare laboratory and clinical outcomes of intracytoplasmic morphologically selected sperm injection (IMSI) and conventional intracytoplasmic sperm injection (ICSI) in couples with repeated implantation failures.MethodsA total of 200 couples with at least two prior unsuccessful ICSI cycles were enrolled: 100 couples were submitted to IMSI and 100 were submitted to routine ICSI. For IMSI, spermatozoa were selected at 8400× magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. For conventional ICSI, spermatozoa were selected at 400× magnification. Clinical outcomes were evaluated between the two groups.ResultsStudy patients were comparable in age, number of treatment failures, aetiology of infertility, percentage of normal form assessed by MSOME (motile sperm organelle morphology examination), semen parameters, total number of oocytes collected, number of mature oocytes collected, total number of embryos transferred and number of high-quality embryos transferred. No statistically significant differences between the two groups were observed with regard to rates of fertilisation, implantation and pregnancy/cycle. Although not statistically significant, rates of miscarriage (IMSI:15.3% vs ICSI:31.7%), ongoing pregnancy (IMSI:22% vs ICSI:13%) and live births (IMSI:21% vs ICSI:12%) showed a trend towards better outcomes in the IMSI group. In addition, analysis of subpopulations with or without male factor showed similar results.ConclusionsOur results suggest that IMSI does not provide a significant improvement in clinical outcome compared to ICSI, at least in couples with repeated implantation failures after conventional ICSI. However, it should be noted that there were clear trends for lower miscarriage rates (≈50% reduced) and higher rates of ongoing pregnancy and live births (both nearly doubled) within the IMSI group. Further confirmation as well as randomized large-scale trials are needed to confirm the beneficial effects of IMSI in couples with poor reproductive prognoses.

Employing a novel video-enhanced contrast-differential interference contrast (VEC-DIC) microscope with a mercury light source, the process of microglial cell death after phagocytosis of zymosan-A was examined for its morphological changes, especially of the nucleus. The materials used were microglia (exp. n = 15) isolated from the newborn rat brain. Zymosan-A was introduced into the chamber on the stage of the microscope where the microglia were being observed. Aggregates of zymosan-A became partially or totally engulfed by the microglia. At ca. 10–30min of phagocytosis, the microglia began to swell, with ballooning of the cell membrane, and then burst. The cell body changes in terms of the circumferential length were: 84.38µm in the control and 141.71µm at the time of bursting (p < 0.05). Those in the area of the cell body were: 36.43µm2 in the control and 47.92µm2 at the time of bursting (p < 0.05). The average dimensional changes of the nucleus (n = 5, where the nuclear changes could be followed) in the control, at the time before bursting, after bursting, and at 10 min after bursting were 7.45, 7.32, 7.25, respectively, and 7.01µm for the long axis, and 6.53, 6.13, 6.81, respectively, and 6.55µm for the short axis. The minimal change in nuclear size together with swelling/dissolution of the plasma membrane suggests that the self-inflicted cell death triggered by zymosan-A, and possibly facilitated by the UV light, might result from necrosis due to self-inflicted effects of autogenous toxic substances. However, the findings were not conclusive since chromatin condensation with cell shrinkage was observed in some cases, suggestive of apoptosis.