In situ observation of elementary growth steps on the surface of protein crystals by laser confocal microscopy

Quasi-liquid layers on ice crystal surfaces are made up of two different phases

“…We attached a confocal system (FV300; Olympus Corporation) to an inverted optical microscope (IX70; Olympus Corporation), as previously explained (6,7). A superluminescent diode (Amonics Ltd.; model ASLD68-050-B-FA: 680 nm) and a He-Ne laser (Melles Griot 05-LHP-991: 633 nm) were used for LCM-DIM and interferometric observations, respectively.…”

“…We and Olympus Engineering Co., Ltd. have developed one such technique-namely, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) (6), which can directly visualize the 0.37-nm-thick elementary steps on ice crystal surfaces with a typical frame rate of 0.1-4 s per frame (7). In this study, we directly visualized surface melting processes on ice crystal surfaces to elucidate the dynamic behavior of QLLs and found that QLLs are made up of two different phases (Fig.…”

Attachment and Detachment Processes of Individual Lysozyme Molecules on a Surface of a Monoclinic Lysozyme Crystal Studied by Fluorescent Single-Molecule Visualization

“…Before and after the single-molecule visualization, the surface morphology of a monoclinic HEWL crystal ( Figure 3a) was observed by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM), by which at first elementary steps on protein crystal surfaces (several nanometers in height) 14 and later elementary steps on ice crystal surfaces (0.37 nm in height) 15 can be visualized with sufficient contrast. From the in situ observation of elementary steps on a monoclinic HEWL crystal by LCM-DIM, we confirmed that elementary steps were not proceeding or receding, proving that a crystal had been maintained in the equilibrium condition during the single-molecule visualization.…”

Protein Crystal Growth Under High Pressure

“…Among the many studies on protein solubilities so far, in situ observation of steps on crystal faces using a laser confocal microscope combined with a differential interference contrast microscope (LCM-DIM (Sazaki et al, 2004)) has been the most powerful method (stepobservation method) for measuring the equilibrium temperatures T e of protein crystals (Van Driessche et al, 2009;Fujiwara et al, 2010). Van Driessche et al reported that this method yielded the highest precision in measurements of T e of tetragonal hen egg-white lysozyme crystals (Van Driessche et al, 2009), and we found it produced the fastest results (Fujiwara et al, 2010).…”

“…However, all R referred in the present review did not depend on the surface area, although different crystals of different size were used. In addition, to confirm the growth mode directly, we observed the surface topography in situ using a reflection type laser confocal microscope combined with a differential interference contrast microscope (LCM-DIM system) (Sazaki et al, 2004) for GI crystals. The 2D nucleation and subsequent lateral growth of the 2D islands were clearly observable.…”