Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Confocal microscopy

Physiological and morphological properties of plant organs can be affected by their prevailing growth microclimate (Sultan, 2000; Niinemets, 2007). A homogeneous light distribution within the crop canopy under diffuse light gives rise to the question whether plant physiological and morphological acclimation occurs. Diffuse light penetrates deeper into the canopy; thus, the lower positioned leaves receive on average a higher light intensity which leads to a higher total nitrogen and chlorophyll content in the canopy, and consequently results in a higher leaf photosynthetic capacity in the lower of the canopy (Li et al., 2014a). Acclimation to diffuse light also includes acclimation of leaf morphology, which affects light interception and, consequently, photosynthesis (Pearcy et al., 2005). Li et al. (2014a) reported that tomato plants grown under diffuse light showed a lower specific leaf area (SLA) which indicates a thicker leaves, as well as a higher leaf area index (LAI) which mainly caused by a greater leaf width. A higher LAI is highly relevant for crop photosynthesis, as long as the fraction of light interception is also increased. For the mature crop under greenhouse condition, which often has a closed canopy, thus, the increased LAI under diffuse light has limited effect on canopy light interception and photosynthesis for mature crop (Li et al., 2014a).

One of the most powerful techniques for introducing contrast into reflected light imaging is differential interference contrast, which allows the visualization of minute elevation differences in surfaces. In the optical configuration (Figure 6(d)), a birefringent prism (also known as a Wollaston or Nomarski prism, depending upon design) is placed in the infinity space just above the objective and a polarizer is installed in the vertical illuminator (similar to polarized light). The prism splits the polarized light wavefronts into two orthogonal polarized beams on their way to the specimen. These perpendicular light beams impact the specimen to create a lateral displacement in regions where surface relief exists. If the surface is completely flat, no features are observed. However, if there is, for example, a small step (see Figure 6(d)) between the two wavefronts, one of the beams must travel a path that is longer and is assigned this path difference. Once the parallel beams have returned to the microscope after passing back through the objective and prism, they pass through a second polarizer (the analyzer) where interference produces an intermediate image where path differences are translated into gray values that can be seen by the eye. Similar to polarized light microscopy, a lambda plate can be positioned beneath the analyzer to shift gray values into colored hues.

Type ofmicroscope

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Fruit and vegetable quality is closely correlated with the pre-harvest growth condition. In open field and conventional clear greenhouses, fruit and vegetables often experience diurnal fluctuations or long-term exposure to direct sunlight, with associated high tissue temperatures. This may result in harvest disorders (i.e., sunburn), and heterogeneity of internal quality properties such as sugar content, tissue firmness, mineral content (Woolf and Ferguson, 2000). Fruit with different temperature histories will also respond differently to postharvest low temperatures (i.e., chilling injury) (Ferguson et al., 1999). The quality problems induced by sunlight exposure could be reduced if plants were grown under diffuse light where less fluctuations in temperature and light intensity occurs, detailed research in this aspect has not been reported so far.

Several techniques are commonly employed to introduce contrast in reflected light microscopy, including darkfield illumination, polarized light, and differential interference contrast. In reflected darkfield microscopy, which is an ideal methodology for exploring the relief in surfaces of materials, wavefronts from the vertical illuminator are directed toward the objective using a specialized mirror assembly that contains an oval opening (see Figure 4 and Figure 6(b)). This light passes through an outer sleeve in the microscope objective and impacts on a ring-shaped concave mirror, which directs the wavefronts at a highly incident angle onto the specimen surface. In cases where the specimen acts as a perfect mirror (in effect, there are no relief features on the surface), there is no light reflected back into the objective from the specimen and the image remains dark. Areas where relief contours exist, however, direct light back into the objective front lens and are observed as being bright features against a very dark background. Note that in darkfield reflected light microscopy, the field and aperture diaphragms in the vertical illuminator should be opened to their widest points so that the light beam illuminating the mirror assembly is not partially blocked.

To investigate the effect of diffuse light on plant processes, many studies have been carried out by comparing plant responses on cloudy and clear days (Zhang et al., 2011; Urban et al., 2012); or by comparing the aftermath of volcanic and anthropogenic emissions (Gu et al., 2003; Mercado et al., 2009). Such type research implies not only a difference in the fraction of diffuse light, but also large differences in light intensity, and the subsequent changes in microclimatic parameters such as air and soil temperature, and vapour pressure deficit (VPD). These changes directly or indirectly influence plant processes. Recently diffuse glass has become available that increases the diffuseness of light without affecting light transmission in the greenhouse (Hemming et al., 2007, 2008, 2014). Studies have reported that such cover materials have a remarkable effect on plant growth and production (Hemming et al., 2007; Li et al., 2014a,b). Thus, the occurrence of diffuse glass not only provide a promising measure for improving horticultural production, but also offers an opportunity to explicitly explore the pure effects of diffuse light on light distribution over the canopy and its direct and indirect effects on crop photosynthesis and plant growth. In this review, we will discuss the effect of diffuse light on plant processes and its application in horticultural production, and subsequently point out the perspectives for further research.

Obviously, diffuse light has great advantageous for plant growth. However, detailed studies about the following aspects that closely related with diffuse light are lacking. Further exploring these aspects will strengthen the scientific understanding of diffuse light modulate plant processes as well as its application for crop production.

In the vertical illuminator, light travels from the light source, usually a 12 volt 50 or 100 watt tungsten-halogen lamp, passes through collector lenses, through the variable aperture iris diaphragm opening and through the opening of a variable and centerable pre-focused field iris diaphragm. The light then strikes a partially silvered plane glass reflector, or strikes a fully silvered periphery of a mirror with elliptical opening for darkfield illumination. The plane glass reflector is partially silvered on the glass side facing the light source and anti-reflection coated on the glass side facing the observation tube in brightfield reflected illumination. Light is thus deflected downward into the objective. The mirrors are tilted at an angle of 45 degrees to the path of the light travelling along the vertical illuminator.

Darkfieldmicroscopy

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A transmitted light microscope will typically be of little use to anyone wanting to examine the structure of metallic samples, the surface of ceramics, integrated circuits, or printed paper documents. As a result, the reflected light microscope has been developed for these purposes. Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers. Much like the fluorescence microscope, in reflected brightfield microscopy the sample is illuminated from above through the objective. The Köhler illumination principle applies in cases where the objective with its pupil plane is also utilized as the condenser.

Objectives for reflected light can be recognized by the Epi or similar inscription on the decorative outer barrel (see Figure 3). They differ from objectives for transmitted light in two ways. Reflected light objectives feature lens surfaces that are particularly well coated with anti-reflection layers to prevent the illuminator light from being reflected towards the eyepiece. Such reflections would be superimposed on the image and have a disturbing effect. The second difference is that these objectives are designed and optically corrected for specimens lacking a coverslip. The vast majority of samples in the materials sciences (where reflected light microscopes are most heavily used) are usually viewed without a cover slip. Therefore, higher numerical aperture objectives require a different optical computation than do transmitted light objectives.

Several modern reflected light illuminators are described as universal illuminators because, with several additional accessories and little or no dismantling, the microscope can easily be switched from one mode of reflected light microscopy to another. Often, reflectors can be removed from the light path altogether in order to permit transmitted light observation. Universal illuminators may include a partially reflecting plane glass surface (the half-mirror) for brightfield (see Figure 4(a)), and a fully silvered reflecting surface with an elliptical, centrally located clear opening for darkfield observation (Figure 4(b)). The best-designed vertical illuminators include collector lenses to gather and control the light, an aperture iris diaphragm and a pre-focused, centerable field diaphragm to permit the desirable Köhler illumination.

Solar light is composed of a diffuse and a direct component. Diffuse light arises from the scattering of light by molecules or larger particles in the atmosphere and comes from many directions simultaneously; direct light arrives in a straight line from the sun without being scattered (Iqbal, 1983). Plants use diffuse light more efficiently than direct light (Gu et al., 2002; Farquhar and Roderick, 2003; Gu et al., 2003; Alton et al., 2007; Mercado et al., 2009; Li et al., 2014a), it arises due to diffuse light creates a more homogeneous light profile in the canopy than direct light. Photosynthetic rate of a single leaf shows a nonlinear response to the light flux density (Marshall and Biscoe, 1980). High light level usually leads to photosynthetic saturation and decrease in light use efficiency (LUE), which often occur under direct light condition. Therefore, the direct light usually wastes photons by concentrating the light resource to only a fraction of all leaves, leading to a less efficient photosynthetic use of light by plant canopies (Gu et al., 2002). Diffuse light, however, effectively avoids the light saturation constraint by more evenly distributing light among all leaves in plant canopies, and leads to a more efficient use of light (Gu et al., 2002).

Polarized reflected light microscopy (Figure 6(c)) is a technique that is suitable for examining surfaces containing structures that alter the state of polarization during the reflection process. For example, structural grains in ore samples and a number of metallic alloys and thin films can be readily examined using this method. In the optical configuration outlined in Figure 6(c), the illuminating wavefronts encounter a polarizer that is placed in the vertical illuminator before the mirror unit that directs light into the objective. The linearly polarized light waves are focused onto the specimen surface and reflected back into the objective. After leaving the objective aperture as a parallel bundle of wavefronts, the light is then projected onto a second polarizer (the analyzer) oriented at 90 degrees with respect to the polarizer. Only the depolarized wavefronts are able to pass through the analyzer to reach the tube lens. An auxiliary lambda plate can also be inserted just prior to the analyzer in the optical train to examine the sign of birefringence (changing gray to color contrast). This method is sometimes referred to as sensitive tint. In cases where objectives of very low magnification are used in reflected polarized light, a rotatable optical plate (termed an Antiflex cap) consisting of a one-quarter wavelength lambda plate is placed on the objective front lens element to block reflections from the objective itself. The Antiflex method is also particularly useful when the specimen has very low reflectivity, such as would be observed in coal samples.

Light distribution in tomato canopy in the conventional clear glasshouse (direct light, A) and diffuse glasshouse (diffuse light, B) on a clear day. Light is more homogeneously distributed under diffuse light (B) compared with direct light (A) where many sunflecks in the middle and lower of the canopy. The photo was taken in Wageningen UR Greenhouse Horticulture, Bleiswijk.

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The image-forming or field set of conjugate planes in reflected light Köhler illumination consists of the field diaphragm, the specimen surface, and the intermediate image plane. Thus, when the field diaphragm is placed in focus at the specimen plane, the image of the light source is significantly removed from focus in order to provide a uniform field of illumination. The field diaphragm controls the size of the illuminated field without affecting the illumination intensity of the area being observed. In practice, the field diaphragm opening size should be as small as possible in order to increase image contrast. Köhler illumination produces even illumination of the specimen field in spite of the uneven illumination intensity generated by most filament-based light sources. When the microscope is properly configured, the rear focal plane of the objective is fully illuminated, providing a field that is uniformly bright from edge to edge. Köhler illumination, in the ideal case, bathes the specimen with a converging set of wavefronts, each arising from separate points on the light source imaged into the condenser aperture. In a properly configured reflected light microscope, the result is optimum image contrast and resolution.

*Correspondence: Tao Li and Qichang Yang, Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agriculture Sciences, Zhongguancun South Street 12, Haidian, 100081 Beijing, China, ltao1985@163.com; yangqichang@caas.cn

Light distribution and absorption is highly dependent on crop architecture (Falster and Westoby, 2003; Zheng et al., 2008; Sarlikioti et al., 2011b). Short and compact canopies may generate substantial leaf overlap and self-shading, therefore decreases the net amount of leaf area exposed to light, and consequently affect canopy light interception (Falster and Westoby, 2003). Plants also vary widely in leaf angle, leaf orientation, internode length, and leaf length to width ratio, these traits have a direct effect on light absorption and photosynthesis (Falster and Westoby, 2003; Sarlikioti et al., 2011b). However, detailed research about plant architecture modulates the effect of diffuse light on light distribution and canopy photosynthesis are lacking. Furthermore, LAI is a predominant factor for canopy light interception, at low LAI mutual shading of leaves within the canopy is small, thus light may readily penetrate deeper into the canopy, which probably decrease the potential effect of diffuse light.

Fluorescencemicroscope

Crop photosynthesis to a large extent correlates with the light profile within the canopy (González-Real et al., 2007; Niinemets, 2007; Sarlikioti et al., 2011a). In the vertical profile of the canopy, light intensity decreases exponentially from top to the bottom of the canopy, as described by the Beer-Lambert–Bouguer law (Chandrasekhar, 1950; Monsi and Saeki, 2005) of which light extinction coefficient can be used to quantify the vertical light distribution in the canopy. Diffuse light exhibits a lower extinction coefficient than direct light (Urban et al., 2012; Li et al., 2014a) although the effect depends on solar position (Morris, 1989). This indicates diffuse light penetrates deeper into the crop canopy. Such phenomenon occurred due to the properties of diffuse light that scatters in many directions and thus causes less shadow, while direct light either concentrates in a beam or casts a shadow in the canopy, which results in the upper leaves being brightly illuminated and lower leaves in deep shade, or strong sunflecks at a given canopy depth. In the horizontal profile of the canopy, diffuse light also results in a more homogeneous light distribution due to less sunflecks occur (Acock et al., 1970; Li et al., 2014a), which plays the most important role for crop photosynthesis enhancement under diffuse light (Li et al., 2014a). A general impression of light distribution in the canopy under direct as well as diffuse light condition has been given in Figure 1. Apart from light distribution, diffuse light also resulted in a lower leaf temperature and less photoinhibition of top leaves (Urban et al., 2012; Li et al., 2014a), which are correlated with the lower light absorption of the top leaves as well as fewer local peaks in light intensity occur under diffuse light, these are also benefit for crop photosynthesis.

In reflected light microscopy, illuminating light reaches the specimen, which may absorb some of the light and reflect some of the light, either in a specular or diffuse manner. Light that is returned upward can be captured by the objective in accordance with the objective's numerical aperture. After entering the objective, light then passes through the partially silvered mirror (or in darkfield, through the elliptical opening). In the case of infinity-corrected objectives, the light emerges from the objective in parallel (from every azimuth) wavefronts projecting an image of the specimen to infinity. The parallel rays enter the tube lens, which forms the specimen image at the plane of the fixed diaphragm opening in the eyepiece (intermediate image plane). It is important to note, that in these reflected light systems, the objective serves a dual function. For light waves on the way down to the specimen, the objective serves as a matching well-corrected (always properly aligned) condenser. Alternatively, for waves reflected by the specimen, the objective serves as an image-forming optical system in the customary role of an objective projecting the image-carrying rays toward the eyepiece. Optimal performance is achieved in reflected light illumination when the instrument is adjusted to produce Köhler illumination (discussed below). A function of Köhler illumination (aside from providing evenly dispersed illumination) is to ensure that the objective will be able to deliver excellent resolution and good contrast even if the source of light is a coiled filament lamp.

Diffuse light improves spatial light distribution in the crop canopy, thereby stimulating crop photosynthesis; the more uniform horizontal light distribution within the canopy plays the most important role for this effect. Diffuse light also lessens the variation of the temporal light distribution at any specific point in the canopy. However, its effect on plant growth depends on the dynamic responses of stomatal conductance to the incident light. Apart from the homogeneous light distribution, diffusing the incident light makes it possible to allow more light in the greenhouse which strongly stimulates crop growth of shade-tolerant pot plants without compromising plant quality. Although the available knowledge have clearly stated the advantageous of diffuse light for crop production, incorporating the seasonal light condition and solar position, plant architecture, crop management practices as well as the post-harvest product quality for further research will strengthen our understanding of the effect of diffuse light on plant processes.

Today, many microscope manufacturers offer advanced models that permit the user to alternate or simultaneously conduct investigations using both vertical and transmitted illumination. A typical microscope configured for both types of illumination is illustrated in Figure 2 (the transmitted light source and optical pathway is not shown in this illustration). The optical pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light (the upper housing in Figure 2). This light next passes through the collector lens and into the vertical illuminator where it is controlled by the aperture and field diaphragms. After passing through the vertical illuminator, the light is then reflected by a beamsplitter (a half mirror or elliptically shaped first-surface mirror) through the objective to illuminate the specimen. Light reflected from the surface of the specimen re-enters the objective and passes into the binocular head where it is directed either to the eyepieces or to a port for photomicrography. Reflected light microscopy is frequently the domain of industrial applications, especially in the rapidly growing semiconductor arena, and thus represents a most important segment of microscopical studies.

In reflected light microscopy, absorption and diffraction of the incident light rays by the specimen often lead to readily discernible variations in the image, from black through various shades of gray, or color if the specimen is colored. Such specimens are known as amplitude specimens and may not require special contrast methods or treatment to make their details visible. Other specimens show so little difference in intensity and/or color that their feature details are extremely difficult to discern and distinguish in brightfield reflected light microscopy. The latter specimens behave much like the phase specimens so familiar in transmitted light work, and are suited for darkfield and reflected light differential interference contrast applications.

Due to the fact that the objective serves a dual purpose (also performing as a condenser) in reflected light microscopy (refer to Figure 6(a)), there is sufficient room to introduce auxiliary components into the infinity space occupied by the parallel bundle of light wavefronts traveling from the objective rear aperture to the tube lens (termed the observation side of the optical train; see Figure 6(a)). In addition, polarizing or filter components can be inserted into the vertical illuminator before light enters the objective (termed the optical train illumination side). Many modern microscopes also provide additional space for components that affect both light paths. This space is usually built as a slot in the objective nosepiece where a slider containing either a filter or polarizer can be easily inserted.

The vertical illuminator should also make provision for the insertion of filters for contrast, digital imaging, and photomicrography, as well as polarizers, analyzers, and compensator plates for polarized light and differential interference contrast (DIC) illumination. In vertical illuminators designed for use with infinity-corrected objectives, the illuminator may also include a tube lens. Affixed to the back end of the vertical illuminator is a lamphouse (Figure 2), which usually contains a tungsten-halogen lamp. For fluorescence work, the lamphouse can be replaced with a fitting containing a mercury burner. The lamp may be powered by the electronics built into the microscope stand, or in fluorescence, by means of an external transformer or power supply.

Light fieldmicroscopy

A typical upright compound reflected light microscope has a viewing tube with two eyepieces (Figure 2) and often a trinocular tube head for mounting a conventional or digital/video camera system (not illustrated). Standard equipment eyepieces are usually of 10x magnification, and most microscopes are equipped with a nosepiece capable of holding four to six objectives. The stage is mechanically controlled with a specimen holder that can be translated in the x- and y- directions and the entire stage unit is capable of precise up and down movement with a coarse and fine focusing mechanism. Built-in light sources range from 20 and 100 watt tungsten-halogen bulbs to higher energy mercury vapor or xenon lamps that are used in fluorescence microscopy. Light passes from the lamphouse through a vertical illuminator interposed above the nosepiece but below the underside of the viewing tube head. The specimen's top surface is upright (usually without a coverslip) on the stage facing the objective, which has been rotated into the microscope's optical axis. The vertical illuminator is horizontally oriented at a 90-degree angle to the optical axis of the microscope and parallel to the table top, with the lamp housing attached to the back of the illuminator. The coarse and fine adjustment knobs raise or lower the stage in large or small increments to bring the specimen into sharp focus.

The effects of diffuse light on crop photosynthesis could strongly differ between winter and summer light conditions. In winter, photosynthesis of the upper leaves is far from light saturation. With the same light intensity at leaf level, upper leaves have a higher rate of photosynthesis than lower leaves. Therefore, deeper penetration of light may have less effect on crop photosynthesis in winter (Sarlikioti et al., 2011b). Furthermore, light interception follows a seasonal pattern with on average, a lower fraction of light intercepted during summer than during winter because of changes in solar elevation (Sarlikioti et al., 2011a). The higher solar elevation in summer months results in an orientation of light rays more perpendicular to the plant canopy, resulting in a higher light penetration and lower interception. Therefore, seasonal variation of light intensity, directional light quality (diffuse or direct light) as well as solar position should be considered when exploring the effect of diffuse light on light distribution and crop photosynthesis.

Row crop systems are commonly used in horticultural and agronomic crops. This system facilitates crop management and allows higher light penetration inside the plant canopy. In this system, a fraction of light reaches the ground floor in the middle of the path (Stewart et al., 2003; Sarlikioti et al., 2011a), the reflection of light by the floor can be reused for photosynthesis. Furthermore, row orientation substantially affects canopy light interception (Borger et al., 2010; Sarlikioti et al., 2011a). These effects may differ between diffuse and direct light conditions.

Inverted reflected light microscope stands incorporate the vertical illuminator within the body of the microscope. Many types of objectives can be used with inverted reflected light microscopes, and all modes of reflected light illumination may be possible: brightfield, darkfield, polarized light, differential interference contrast, and fluorescence. Some of the instruments include a magnification changer for zooming in on the image, contrast filters, and a variety of reticules. Because an inverted microscope is a favorite instrument for metallographers, it is often referred to as a metallograph. Manufacturers are largely migrating to using infinity-corrected optics in reflected light microscopes, but there are still thousands of fixed tube length microscopes in use with objectives corrected for a tube length between 160 and 210 millimeters.

Opticalmicroscope

Plants use diffuse light more efficiently than direct light, which is well established due to diffuse light penetrates deeper into the canopy and photosynthetic rate of a single leaf shows a non-linear response to the light flux density. Diffuse light also results in a more even horizontal and temporal light distribution in the canopy, which plays substantial role for crop photosynthesis enhancement as well as production improvement. Here we show some of the recent findings about the effect of diffuse light on light distribution over the canopy and its direct and indirect effects on crop photosynthesis and plant growth, and suggest some perspectives for further research which could strengthen the scientific understanding of diffuse light modulate plant processes and its application in horticultural production.

The resolving power in reflected light is based on the same relationship between the wavelength of light and numerical aperture (the Abbe equation) as in transmitted light. Optical performance is achieved in reflected light illumination when the instrument is adjusted to operate under Köhler illumination. A function of Köhler illumination (aside from providing evenly dispersed illumination) is to ensure that the objective will be able to deliver excellent resolution and good contrast even if the source of light is a coiled filament lamp. In many cases, modern reflected light microscopes may also be operated using transmitted light because the parfocal length is maintained in all objectives.

On the inverted stand (similar in basic construction to the inverted tissue culture style microscope frames commonly employed in biology), the specimen is placed on the stage with its surface of interest facing downward. The primary advantage of this design is that samples can be easily examined when they are far too large to fit into the confines of an upright microscope (such as large rock samples and industrial materials). Also, only the side of the specimen facing the objectives need be perfectly flat. The objectives are mounted on a nosepiece under the stage with their front lenses facing upward towards the specimen and focusing is accomplished either by moving the nosepiece or the entire stage up and down.

Even in northern countries, there are periods in summer with too high light levels for many shade-tolerant pot plants such as anthurium, bromeliads, and orchids. When excessive light energy is being absorbed by the light harvesting antennae at a rate which surpasses the capacity for photochemical and non-photochemical energy dissipation, this may lead to photoinhibition or photo-damage (Long and Humphries, 1994). In the long term, this could result in discoloring of leaves or even necrosis. Light damage occurs mostly as a result of prolonged exposure to excessive peaks in light intensity (Asada, 1999; Niyogi, 1999; Kasahara et al., 2002). Consequently, growers regularly apply shading in commercial production of many shade-tolerant pot plants in summer by closing a screen or having white wash on the greenhouse cover. However, shading often carries a penalty on potential crop growth as it is positively related to the amount of light that can be captured, which consequently reduces the LUE in the greenhouse production systems. When diffusing the incident light through cover materials, light in the greenhouse is more homogeneously distributed with less sunflecks, which decreases the extent of photoinhibition as well as local peaks in leaf temperature when global radiation is high (Li et al., 2014a). Therefore, the problem of discoloring of leaves or necrosis in shade-tolerant pot plants under relatively high light could be less when cultivated under diffuse light condition (Li et al., 2014b). Studies have suggested that increasing daily light integral under diffuse light not only accelerates plant growth but also improves plant ornamental quality with more compact plants (Li et al., 2014b; Marcelis et al., 2014). This may substantially contribute to the improvement of horticultural production.

In nature, temporal light distribution in the canopy is characterized by alternating periods of relatively high light followed by periods of background low light at a given point (sunflecks). Under these circumstances, a large fraction of CO2 assimilation may occur under transient light conditions. Stomata regulate carbon uptake of a leaf. In response to fluctuating light, stomata exhibit a dynamic response that is slower than the response of photosynthesis and fluctuating light itself, which may limit the CO2 assimilation under fluctuating light conditions (Pearcy et al., 2004; Lawson and Blatt, 2014). In greenhouses, the shadow and sunflecks generated by overstory leaves, leaf movement, greenhouse construction parts as well as equipment may exacerbate the variation of temporal light distribution. This may substantially limit crop photosynthesis compared to constant light intensities (Pearcy, 1990; Way and Pearcy, 2012). This variation in light intensity can be minimized when the incident light is made diffuse, which would consequently lead to less limitation on leaf photosynthesis, thus improving the canopy LUE (Li et al., 2014b).

In reflected light Köhler illumination (illustrated schematically in Figure 5), an image of the light source is focused by the collector lens onto the aperture iris diaphragm located in the vertical illuminator. This diaphragm shares a conjugate plane with the rear aperture of the objective and the lamp filament, and therefore, determines the illuminated field aperture size. Together, the light source, vertical illuminator aperture diaphragm, and objective rear focal plane (pupil) form the illumination set of conjugate planes. Unlike the situation in transmitted light microscopy, the aperture iris and light source are imaged onto the objective (acting as a condenser) rear aperture plane, rather than being physically located at this position. As an added benefit to this configuration, all obstructions (such as iris diaphragms) are removed from the light path. Opening or closing the aperture diaphragm is used to control stray light and regulate the intensity (numerical aperture) of illumination without altering the size of the illuminated field. In the image, adjustment of the aperture diaphragm affects brightness and contrast.

Stomatal responses to dynamic light vary dramatically among species, from virtually no response to rapid stomatal responses, thereby resulting in different consequences for instantaneous leaf photosynthesis (Knapp and Smith, 1990; Vico et al., 2011), which may subsequently modulate the effect of diffuse light on canopy LUE. Li (2015) have tested the responses of two anthurium cultivars which have distinct stomatal properties to diffuse light. In cultivars where stomata respond strongly to fluctuations of photosynthetic photon flux density (PPFD), transient rates of photosynthesis and subsequently LUE increased under diffuse light in which stomatal conductance becomes relatively constant and less limiting for photosynthesis. For cultivars with relatively insensitive stomata to the fluctuations of PPFD, the effect of the homogeneous temporal distribution of PPFD on LUE was non-existing. In this context, additional to benefits of diffuse light associated with improved spatial light distribution, the stimulating effect of diffuse light on crop LUE can also depend on the dynamic response of stomatal conductance to incident PPFD at leaf level.

This work was supported by the National High-tech R&D Program of China (863 Program) under contract number 2013AA102407.

The range of specimens falling into this category is enormous and includes most metals, ores, ceramics, many polymers, semiconductors (unprocessed silicon, wafers, and integrated circuits), slag, coal, plastics, paint, paper, wood, leather, glass inclusions, and a wide variety of specialized materials. Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection. As mentioned above, such illumination is most often referred to as episcopic illumination, epi-illumination, or vertical illumination (essentially originating from above), in contrast to diascopic (transmitted) illumination that passes through a specimen. Several reflected light specimens are presented in Figure 1. The surface of an integrated circuit is shown using reflected light differential interference contrast (DIC) in Figure 1(a), while the jewel bearing of a watch mechanism captured in brightfield is presented in Figure 1(b). Darkfield is another useful reflected light technique, as evidenced by the image revealing surface structure of a superconducting wire cable in Figure 1(c). Finally, a magnetic thin film (Figure 1(d)) can be imaged using polarized reflected light microscopy to examine surface defects (blisters) that affect the homogeneity of the film.

Measuring leaf photosynthesis is the basis for estimating canopy photosynthesis. Conventionally, only the adaxial side of the leaf is illuminated by the light source when measuring single leaf photosynthesis, this might result in minor error in estimating the canopy photosynthesis under diffuse light. This is because diffuseness of light may affect the fraction of light on the abaxial leaf surface, while the abaxial surface have a different photosynthesis light response curve than adaxial surface (Paradiso and Marcelis, 2012). Therefore, measurements of light absorption and photosynthesis light response curves on both the adaxial and abaxial side of leaves in the canopy in combination with functional–structural plant modeling might help to estimate these effects.

In a reflected light microscope vertical illuminator, the light source is positioned so that the tungsten-halogen lamp filament is located near the principal focal point of the collector lens. In Köhler illumination, the lamp collector lens serves the function of a dramatically enlarged secondary light source to enhance overall illumination. One of the primary requirements of Köhler illumination is that an image of the lamp filament must ultimately be projected onto the rear focal plane of the objective, which also doubles as the (often high numerical aperture) condenser during excitation in reflected light illumination. The light source should ideally fill the entire objective aperture to both maximize the intensity of radiation and to produce an evenly illuminated field. In many cases, a ground glass filter is placed into the vertical illuminator between the lamphouse and the neutral density filters in order to increase the uniformity of illumination. However, because diffusion filters also reduce the level of illumination, they should be avoided whenever possible.