Dynamic Phase Microscopy Research Articles

Using dynamic phase microscopy for studies of the neuron cytoplasm

Using laser interference microscopy (LIM), an approach to study the state of the plasma membrane (PM) and membrane region of the neuron cytoplasm (vertebrates and invertebrates) has been developed. Changes of the state of the plasma membrane and cytoplasm of neurons at rest and modifying their functioning (using the neurotransmitter acetylcholine) were studied. A method for evaluation of the kinetics of the changes in the plasma membrane and cytoplasm of neurons in different parts of the cell has been developed. Regular processes in the plasma membrane and cytoplasm of neurons, with their frequency depending on the functional state of the neurons, have been found. It was suggested that the fluctuations depend on the changes in the relief of the plasma membrane, restructuring of the membrane cytoskeleton, and movement of intracellular vesicles in the neuron cytoplasm.

Dynamic phase microscopy reveals periodic oscillations of endoplasmic reticulum during network formation.

Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6-2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6-2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.

Identification of two discrete states of energized mitochondria: Experiments on single mitochondria

Dynamic phase microscopy was used to measure the refractivity of a single mitochondrion. Our previous studies showed that application of an electric potential to artificial and natural mitochondrial membranes sharply increases their refractivity. Under the conditions of proton pump activity, the refractivity of a single mitochondrion is 2 to 4 times higher than an average refractivity of deenergized mitochondria. This study demonstrates that the membrane potential of energized mitochondria varies depending on environmental conditions and is controlled by the mitochondrial osmoregulation system. The refractivity of energized mitochondria, i.e., the difference between the refraction indexes of a single mitochondrion and the medium, is 0.02 ± 0.01, i.e., several times lower than that of the energized mitochondria whose membranes bear an electric charge. Earlier it was shown that refractivity of model multilayer systems formed from purified natural lecithin depends linearly on the electric field strength. These data point to a relationship between the refractivity of a single mitochondrion and the membrane potential generated during operation of the proton pump. Under normal conditions (250 mOsm), the mitochondrion behaves as a dynamic system oscillating on a minute scale between two functional states with different values of the refractivity index and different membrane potentials. The transition time is 10–30 s; the lifetime of both states is several minutes. The histograms reflecting the distribution of refractivities of single energized mitochondria within a population (n = 20–30) revealed the presence of two independent peaks (fractions II and III) with average refractivity values of 0.05 ± 0.01 and 0.09 ± 0.01, respectively; these fractions correspond to two long-lived states of mitochondria. However, under hypotonic conditions (120 mOsm), only one (“static”) state was identified, in which oscillations were absent and the refractivity of the overall mitochondrial population does not exceed 0.05 ± 0.01 (fraction II). Studies on mitoplast showed that values of refractivity are related to the inner mitochondrial membrane. It is inferred from these data that there exist two discrete states of mitochondria. Analysis of low-amplitude fluctuations of the refractivity of single mitochondria revealed the presence of frequency components at 1–3 Hz, presumably generated in response to non-uniform functioning of mitochondrial proton pumps. It is suggested that frequency components at 1.8-2.6 Hz are more characteristic of the ATPase pump, while the 1–1.3 Hz frequencies predominate during the functioning of respiratory proton pumps.

Dynamic phase microscopy: is a 'dialogue' with the cell possible?

The metabolic component of refractivity is considered as an example of a quantifying parameter for the 'vitality' of cells and organelles. It is shown that intracellular processes can be studied by the dynamic phase microscopy method, whichprovides real-time information in the form of local changes in the optical path difference. Preliminary results demonstrate the possibility of localizing a 'signal' and obtaining information under the signal – structure – function scheme and show prospects for conducting a 'dialogue' with a cell by real-time registering the way in which it responds to changes in external conditions. The mechanism underlying the dependence of refractivity on metabolic states is explained by the contributionof bound water molecules.

Research on the early stages of spore germination in Bacillus licheniformis using dynamic phase microscopy

The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is > or = 80 nm, 40-50 nm, and < or = 0 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40 degrees C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.

Dynamic phase microscopy, a new method to detect viable and killed spores and to estimate the heterogeneity of spore populations

One of the challenging tasks in monitoring studies is to estimate heterogeneity of microbial populations by the physiological state and potential viability of individual cells, especially with regard of their ability to withstand various environmental assaults. Previously, we described some approaches based on electron microscopy methods to discriminate vegetative, dormant, and dead cells in both aged microbial cultures and environmental samples, including permafrost. We propose to extend the arsenal of microscopy methods for monitoring studies by a new non-invasive and informative method – dynamic phase microscopy (DPM). The substantial advantage of DPM is that it gives quantitative (digitized) data of undestroyed (living) microscopic objects, exemplified in our work by Bacillus licheniformis spores. Using DPM made it possible to record interference images of objects (spores) and to produce picture of their “phase thickness” (PT) that is the optical path difference in nm. Thus, it was demonstrated the remarkable difference in the PT of spores at different physiological states: dormant, germinating, and heat-killed spores had PT values of 80, 40–50, and 20 nm, respectively. The other found criterion to distinguish between spores was the PT fluctuations. In contrast to dormant and killed spores, the PT of germinating spores oscillated with amplitude of up to 7 nm, with typical frequencies of 1.3 and 3.4 Hz. A combination of the recorded PT values and PT fluctuations gave a key to detect viable and dead cells. Under the conditions that did not support germination (the lack of nutrients), we were able to follow the response of a single dormant spore and a spore population to heating from 25 °C to 70 °C. Thus, a very small temperature change (from 40 °C to 42 °C) under conditions non-favorable for germination, caused a drastic decrease in the spores’ PT; the second drop in the PT values was observed during heating from 60 °C to 70 °C. These changes were reversible: after cessation of heating, PT values became similar to dormant spores. So, DPM allowed us to track the first, reversible stage of activation, when a spore maintains the attributes of the dormant state. Under the conditions that favor germination (in the presence of nutrients), irreversible changes in the PT and spore diameter, d, were detectable in a single germinating spore and spore population. In addition, DPM allowed an easy estimation of the heterogeneity of spore populations. It is a great advantage of DPM that it makes possible to reveal the ability of spores to respond to various stimuli with or without further germination and outgrowth – the salient feature of a living cell. DPM may have a high potential in general microbiology and astrobiology, enabling to: (1) estimate the heterogeneity of spore populations either under standard conditions and subjected to solar radiation and simulated extraterrestrial factors; (2) to track a response of spores to changing conditions at the early germination stage, even if they do not enter further outgrowth; (3) to develop some approaches for monitoring studies and appraisal of the physiological state of dormant cells in situ, in samples of dry soils, permafrost, etc. regarded as models for searching life beyond the Earth.

A dynamic phase microscopic study of optical characteristics of individual chloroplasts

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h( x, y, t) ≈∫ n( x, y, z, t)d z, an integral refractive index projection of the medium, n( x, y, z, t), in optically transparent biological specimens at high spatial and temporal resolutions. In this study, DPM was used for the analysis of fluctuations in the optical characteristics of individual bean chloroplasts in various metabolic states. A “phase image” of an individual chloroplast, which represents a three-dimensional plot of the “phase height”, was obtained for the first time, and the frequency spectra of the fluctuations of h( x, y, t) were investigated. The fluctuation patterns, i.e., the intensity and the frequency spectra of phase height fluctuations in bean chloroplasts (Class B) were found to depend on their metabolic state. Under conditions of noncyclic (or pseudocyclic) electron transport, the fluctuations displayed characteristic frequencies in the range of 0.25–0.6 Hz and were space–time-correlated in the chloroplast domains with the cross sizes of ∼2 μm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 μM). A stronger (in comparison with 20 μM valinomycin) effect of 20 μM nigericin suggests that the light-induced generation of the transmembrane pH difference (ΔpH) makes the main contribution to the increment of space-correlated fluctuations of h( x, y, t). Studies of chloroplasts incubated in media of various osmolarity (50–500 mM sucrose) have shown that structural changes in thylakoids are among other factors responsible for phase height fluctuations.

Superresolution at the singularities of phase images

Measurements performed with an Airyscan laser phase microscope showed the possibility of localisation of structural elements of a surface with a large gradient of the profile height and linear dimensions as small as 25 — 40 nm, which is 10 — 15 times lower than the Rayleigh criterion. The width of the structural elements was estimated from the extent of a region of the enhanced intensity of phase fluctuations. A metallised CD substrate with the known surface structure was used as a test object. Fluctuations of the optical path difference, whose intensity increased at steep profile slopes, were measured by periodically scanning the surface profile. It is shown that the extent of the region of enhanced intensity of phase fluctuations is close to the measured width of the steep profile slope. The intensity of fluctuations increased proportionally to the square of the height gradient, while their spatial extent decreased noticeably with increasing the objective aperture. These measurements explain a high sensitivity of the dynamic phase microscopy to fluctuations of the optical path difference in the regions of high gradient of the refractive index. The increase in the intensity at the slopes of the phase height profile and the spatial — temporal correlation of fluctuations were observed in mitochondria and other biological objects.

A study of demyelination of nerve fibers using dynamic phase contrast microscopy.

Dynamic phase microscopy was used for evaluation of changes in myelinated axon segment in the paranodal region of nerve fibers during demyelination. Normally paranodal myelin sheath is characterized by regular oscillations of the optical path difference with frequences of 4.2 and 6.7 Hz. Demyelination decreased the amplitude and conduction velocity in nerve fibers and shifted the characteristic frequencies of optical path difference oscillations to 2.8, 3.2, and 11 Hz. These shifts of optical path difference frequencies probably resulted from disturbances in the state of charged phospholipids and a decrease in the level of bound Ca(2+)during demyelination of nerve fiber.