Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period.2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms).3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change.4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation.5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics.6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity.7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant.8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.     

Imaging by Delayed Light Emission (Phytoluminography) as a Method for Detecting Damage to the Photosynthetic System

An improved apparatus for obtaining luminescence (delayed light emission) images of plants is described. It consists of a phosphoroscope equipped with an imaging lens and an electronic image intensifier. It is also equipped with light-sources for obtaining images with reflected light and fluorescence light. It is shown that damage to the photosynthetic system caused by virus, insects, high or low temperature, ultraviolet radiation, or herbicide, and also chioroplast senescence as part of a normal developmental process, can be followed by this non-destructive method. In many cases changes which are not visible in fluorescence images are clearly seen in luminescence images.     

Comparative experimental and theoretical study of the dependence of parameters of curved induction of fluorescence and millisecond delayed luminescence on dark adaptation time

The dependences of G = (Fm - Fs)/Fm parameter for slow fluorescence induction and delayed luminescence induction curves on adaptation time t(ad) were obtained both in experiment, for the leaves of Hibiscus rosa chinensis in vivo, and in a theoretical study with the help of the theoretical model developed earlier. In both cases, the G(t(ad)) dependences have the form of curves with saturation, which are well described by G = A(1 - exp(-(t(ad)/T0)). Both in experiment and in theoretical calculations, G values for delayed luminescence were larger than for fluorescence. The ratio of saturation times for G(t(ad)) for fluorescence and delayed luminescence was 1.40 for experiment and 1.46 for theoretical calculations. These results suggest that delayed luminescence induction is more sensitive to changes in the state of plant than the slow fluorescence induction.     

Delayed fluorescence from Rhodopseudomonas viridis following single flashes.

Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studies using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P+) of the primary electron-donor (P) and the photoreduced form (X MINUS) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P+ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P+X minus, and (c) upon transfer of an electron from X minus to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P+ by cytochromes and the oxidation of X minus by Y, the decay kinetics of delayed fluorescence are identical with those of P+X minus, as measured from optical absorbance changes. The main decay route for P+X minus under these conditions has a rate-constant of approximately 10-3-s-minus 1. In contrase, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P+X minus returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s-minus 1, at 295 degrees K and pH 7.8. The decay kinetics of P+X minus and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal mol-mol- minus 1. We conclude that the main decay route involves tunneling of an electron from X minus to P+, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P+X minus results in increases of both the energy and the entropy of the system, by 16.6 kcal-mol- minus 1 and 8.8 cal-mol- minus 1-deg- minus 1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.     

Precursors to microsecond delayed luminescence in oxygenevolving and inhibited chloroplasts

We have investigated submillisecond delayed luminescence in spinach chloroplasts under a variety of conditions. In Tris-washed chloroplasts, which are inhibited on the oxidizing side of P-680, the delayed light emission in the 7–200 μs time-range decayed with biphasic behavior. In fully dark-adapted samples illuminated by a single saturating laser pulse, the fast phase of delayed luminescence followed a nearly identical pH-dependent time-course as that observed optically and by ESR for P⁺-680 reduction, thus verifying the recombination hypothesis for the origin of delayed light. The observed slower phase of delayed luminescence was also pH dependent, but unlike the fast phase, could not be ascribed to specific electron transfer events of PS II. This phase could be rationalized by a heterogeneity in the population of P-680. While kinetic parameters were found to be insensitive to changes in ionic strength, the overall luminescence intensity was quite sensitive to the electrical parameters, thus indicating the role of ionic strength and local charges in delayed luminescence modulation. A similar series of experiments was performed on untreated chloroplasts. The pH-dependent delayed luminescence behavior in both untreated chloroplasts and Tris-washed chloroplasts was similar despite significantly faster kinetics associated with the reduction of P⁺-680 by the secondary PS II electron donor, Z, in the former preparation (e.g., Van Best, J.A. and Mathis, P. (1978) Biochim. Biophys. Acta 503, 178–188). Thus, it was concluded that, in untreated samples, microsecond delayed luminescence emanates primarily from centers which are not competent in oxygen evolution. The nearly identical delayed luminescence intensity in untreated chloroplasts and in Tris-washed chloroplasts was rationalized by a model which predicts modulations in delayed luminescence yield by the exciton-quenching effect of P⁺-680. Computer simulations demonstrate the feasibility of this model. The previously documented flash oscillations in microsecond delayed luminescence intensity in untreated chloroplasts (Bowes, J.M. and Crofts, A.R. (1979) Biochim. Biophys. Acta 547, 336–346), which we readily observed, were attributed to alterations in delayed luminescence yield (in nonfunctional centers) by variations in charge density stored at the oxygen-evolving complex of functional centers. Taken together, our results emphasize the dependence of delayed luminescence kinetics upon electron-transfer kinetics and the dependence of delayed luminescence amplitude upon the photochemical parameters, the exciton yield and the emission yield.     

Induction kinetics of delayed fluorescence of sun and shade leaves ofFagus sylvatica in the ms-range

Summary Induction kinetics of luminescence (=delayed chlorophyll fluorescence or delayed light emission) were measured with sun and shade leaves of a tall beech tree (Fagus sylvatica pendula, weeping beech). The kinetics detected in the ms-range are contrasted for the upper and the lower leaf side. The influence of the following parameters is demonstrated: time of dark-adaptation prior to the measurement, intensity of the excitation light and photoinhibitory treatment. The effects are discussed with respect to chlorophyll concentration, absorption of the excitation light, reabsorption of the luminescence and photosynthetic activity of the leaf tissue. It is shown that the luminescence signal and its kinetic are determined mainly by the properties of the mesophyll parenchyma facing the detector. Thus the more densely packed palisade parenchyma at the upper leaf side exhibits a lower luminescence and a slower kinetic than the spongy parenchyma at the lower leaf side, which is characterized by many aerial interspaces. Our study shows that luminescence kinetics can be applied to interpret the physiological state of a specific leaf tissue. They may serve as an indicator of disorders in the photosynthetic function.     

External electric field effects on prompt and delayed fluorescence in chloroplasts

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.     

Study of triple-singlet energy transfer in an enzyme-dye complex using optical detection of magnetic resonance.

We have made optical detection of magnetic resonance (ODMR) measurements on the enzyme alpha-chymotrypsin, as well as on its complex with the dye, proflavin. Evidence that triplet-singlet energy transfer occurs in the complex is provided by the observation of characteristic tryptophan ODMR signals while monitoring the delayed fluorescence of the dye. The luminescence decay kinetics of the complex indicates that nontrivial triplet-singlet transfer originates from several (at least three) tryptophan residues of the enzyme. ODMR sensitivity can be enhanced by coupling the sublevels of a weakly radiative triplet state to a fluorescent dye which satisfies F?rster's (F?rster, T. (1948), Ann. Phys. (Leipzig) 2, 55; (1965), in Modern Quantum Chemistry, Istanbul Lectures, Part III, Sinanoglu, O., Ed., New York, N.Y., Academic Press, p 93) conditions for energy transfer.     

Computer aided fluorescence imaging of photosynthetic systems

A fluorescence video imaging system utilizing relatively inexpensive commercial components is described. The instrument utilizes a black and white CCD video camera detector, a commercial video imaging board and a IBM-AT compatible computer. The color output of the imaging board greatly aids in the users ability to visually discriminate areas of interest in the video field. Software development that enables the user to capture kinetic traces in real time from the video images is also described. The system is used to monitor fluorescence from photosynthetic systems. The usefulness of the system in screening for photosynthetic mutants is also demonstrated. The cost of the system can be kept below $12,000.Abbreviations CCD charge-coupled device - DCMU diuron, 3-[3,4-Dichlorophenyl]1,1-dimethylurea - AGC automatic gain control - LUT look-up table - AOI area of interest - CPU central processing unit - RAM random access memory - ADC analog-to-digital converter - FVIPS fluorescence video image processing software - I/O input/output - F₀ dark-level fluorescence - OIDPSMT characteristic transient components, where O is dark level, I is intermediary peak, D is dip, P is peak of fast transient, S is quasi-steady state level, M is second maximum, T is terminal level     

An instrument capable of imaging chlorophyll a fluorescence from intact leaves at very low irradiance and at cellular and subcellular levels of organization

An instrument capable of imaging chlorophyll a fluorescence, from intact leaves, and generating images of widely used fluorescence parameters is described. This instrument, which is based around a fluorescence microscope and a Peltier-cooled charge-coupled device (CCD) camera, differs from those described previously in two important ways. First, the instrument has a large dynamic range and is capable of generating images of chlorophyll a fluorescence at levels of incident irradiance as low as 0.1 μmol m^?2 s^?1. Secondly, chlorophyll fluorescence, and consequently photosynthetic performance, can be resolved down to the level of individual cells and chloroplasts. Control of the instrument, as well as image capture, manipulation, analysis and presentation, are executed through an integrated computer application, developed specifically for the task. Possible applications for this instrument include detection of early and differential responses to environmental stimuli, including various types of stress. Images illustrating the instrument's capabilities are presented.     

Real-time multi-wavelength fluorescence imaging of living cells

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.     

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores was studied at room temperature and under intermittent illuminations. The decay of delayed fluorescence was constituted of two components; a fast component decayed with a half time of about 8 ms, a slow one decayed in parallel with the reduction of photooxidized bacteriochlorophyll (P+) with a half time of 100-200 ms. The biphasic decay of delayed fluorescence indicated that a rapid equilibrium was established between the primary electron acceptor and the secondary acceptor. In the presence of o-phenanthroline, the time course of the decay of delayed fluorescence was identical with that of the reduction of P+ in reaction center-rich subchromatophore particles, although they did not necessarily coincide with each other in "intact" chromatophores. The intensity of the slow component was increased and the decay was accelerated at basic pH values. Reagents that dissipate the proton gradient across the chromatophore membranes such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and nigericin accelerated the decay of the slow component. These effects are probably resulting from changes in internal pH of chromatophore vesicles. Reagents that dissipate the membrane potential such as CCCP and valinomycin decreased the intensity.     

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence measurement by a streak camera in a single-photon-counting mode

We describe here a recently developed fluorescence measurement system that uses a streak camera to detect fluorescence decay in a single photon-counting mode. This system allows for easy measurements of various samples and provides 2D images of fluorescence in the wavelength and time domains. The great advantage of the system is that the data can be handled with ease; furthermore, the data are amenable to detailed analysis. We describe the picosecond kinetics of fluorescence in spinach Photosystem (PS) II particles at 4–77 K as a typical experimental example. Through the global analysis of the data, we have identified a new fluorescence band (F689) in addition to the already established F680, F685, and F695 emission bands. The blue shift of the steady-state fluorescence spectrum upon cooling below 77 K can be interpreted as an increase of the shorter-wavelength fluorescence, especially F689, due to the slowdown of the excitation energy transfer process. The F685 and F695 bands seem to be thermally equilibrated at 77 K but not at 4 K. The simple and efficient photon accumulation feature of the system allows us to measure fluorescence from leaves, solutions, single colonies, and even single cells. The 2D fluorescence images obtained by this system are presented for isolated spinach PS II particles, intact leaves of Arabidopsis thaliana, the PS I super-complex of a marine centric diatom, Chaetoceros gracilis, isolated membranes of a purple photosynthetic bacterium, Acidiphilium rubrum, which contains Zn-BChl a, and a coral that contains a green fluorescent protein and an algal endosymbiont, Zooxanthella.     

Transient peaks in the delayed luminescence from Scenedesmus obtusiusculus induced by phosphorus starvation and carbon dioxide deficiency

Cells of the unicellular green alga Scenedesmus obtusiusculus (Chod.) were starved of phosphorus for 24, 48, 72 and 96 h, and the decay kinetics of the delayed luminescence from the differently starved cells was monitored for several minutes. Cells starved for 24 h showed similar delayed luminescence decay kinetics and accumulated output of photons as control cells after excitation with white light. Two transient peaks (with several components) in the decay kinetics of delayed luminescence were observed after 48 h of phosphorus starvation but not after 72 or 96 h. The amplitude of the transient peaks varied depending on the length of the excitation period with white light and on the length of the dark period preceding light excitation. High CO₂ availability induced no transient peak, whereas low CO₂ availability induced a high transient peak. Transient peaks could not be induced by excitation with light of 660 or 680 nm and only a single transient peak developed using 700 nm light. The kinetics of the delayed luminescence was changed, and the accumulated output of photons was decreased when the pH of the medium was changed from 7.2 to 9.5, both in cells starved for phosphorus for 96 h and in controls. The data indicate that a complicated metabolic pattern is involved in the mechanisms giving rise to the observed transient peaks in the delayed luminescence. The main factors may be a reduction in the translocation of trioses from chloroplasts, a concomitant reduction in Calvin cycle activities and changes in the amount of ATP and reducing agents available.     

Delayed fluorescence in photosynthesis

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon—delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.     

Mechanisms of chlorophyll fluorescence revisited: Prompt or delayed emission from photosystem II with closed reaction centers?

This paper proposes a model which correlates the exciton decay kinetics observed in picosecond fluorescence studies with the primary processes of charge separation in the reaction center of photosystem II. We conclude that the experimental results from green algae and chloroplasts from higher plants are inconsistent with the concept that delayed luminescence after charge recombination should account for the long-lived (approx. 2 ns) fluorescence decay component of closed photosystem II centers. Instead, we show that the experimental data are in agreement with a model in which the long-lived fluorescence is also prompt fluorescence. The model suggests furthermore that the rate constant of primary charge separation is regulated by the oxidation state of the quinone acceptor Q_A.     

High intensity solid-state UV source for time-gated luminescence microscopy.

BACKGROUND: The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the time-domain can selectively resolve probes with long fluorescence lifetimes (tau > 100 micros) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime. METHODS: A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a high-power (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime >500 micros. RESULTS: BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to autofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 mus, the SNR was improved to 12.8:1, a 25-fold improvement. CONCLUSION: In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture.     

Characteristics of the intrinsic luminescence of Actinomyces olivocinereus--producer of heliomycin]

Some characteristics of UV-induced luminescence were studied with Actinomyces olivocinereus producing the antibiotic heliomycin. The luminescence of the growth medium was found to be caused not by heliomycin, but by some other factors. The luminescence of heliomycin in the colonies was quenched as a result of its screening with melanin pigments located in a layer between the aerial and substrate mycelium.