- Email: [email protected]
Phase Imaging in Plant Cells and Tissues
CHAPTER 4
Phase Imaging in Plant Cells and Tissues Vassilios Sarafis Botany School University of Melbourne, Vic, Australia Mathematics and Physics School, University of Queensland, Brisbane St Lucia, QLD, Australia Biomedical Science and Engineering School, University of Adelaide, SA, Australia Birla Institute of Technology and Science, Hyderabad, Andhra Pradesh, India
Editor: Zeev Zalevsky
Phase imaging in plant cells and tissues depends on the boundaries between places where concentration of water is different or there is a boundary between water and oil containing compartments. This chapter has listed 10 microscopy methods to make the boundaries to be visible to the human eye so that they can be well studied. These methods can be applied for transparent specimens of different characteristics. The out of focus imaging is the first and the earliest method that produces contrast at the boundary of the specimen using the out of focus approach. The dark field microscopy creates dark background around the specimen using the elimination of the unscattered beam from the image of the specimen. The phase contrast techniques, presented Chapter 1, allow a living specimen to be studied due to its exclusion of staining showing the boundaries where refractive index changes. There are also other types of contrast microscopy. One is to use Becke line test to create contrast between the boundaries of specimens. Differential contrast microscopy (DIC), presented in Chapter 2, is another type of contrast microscopy. It uses the interference of dual polarizations to form visible images of specimens due to the phase difference. Also, the Hoffman modulation contrast (HMC) microscope is able to vary the contrast of different regions within a specimen using its phase gradients. The adaptive optics microscopy, on the other hand, uses adaptive optics elements such as deformable membrane mirror (DMM) or spatial light modulators to optimize the image quality of the specimen, usually in fluorescence. The interferometric microscopy, also uses phase contrast technique with the addition of a spatial light interference microscopy (SLIM) module to capture the image of a specimen. Biomedical Optical Phase Microscopy and Nanoscopy © 2013 Elsevier Inc. All rights reserved.
53
54
Chapter 4
The second harmonic imaging microscopy (SHIM) obtains the half wavelength signal which is due to the nonlinear optical effect of the specimen for imaging. The final part of this chapter shows the flowing image in the plants using the magnetic resonance imaging (MRI) as well as static water and lipids.
4.1 Out of Focus Imaging The earliest method for observing phase variations is imaging at various focal positions. This earliest method is known as out of focus contrast. Let us consider an object in focus. It is utterly transparent but has a boundary separating it from water with the same refractive indices in and out of the boundary. If in true focus we shall see nothing in a transparent unstained specimen. Just out of focus it will have contrast which reverses as we go through true focus to the other side off the true focus. The contrast is due to partial coherence of the light waves originating from parts of the object prior to and after the object being observed [1]. Using Adobe Photoshop, it is possible to increase the contrast of such images considerably. Such a method has been used by some Indian botanist scientists to observe enhanced contrast in pollen grains mounted in water. In that case, there is a difference in concentration of materials in the pollen grain and the outside which is pure water and this enhances the contrast due to refraction at the boundaries as well.
4.2 Dark Field An excellent method for visualizing boundaries is dark field. In this method, direct light from the microscope condenser is excluded from entering the objective [2,3]. A high aperture may be used for illumination up to 1.42 in commercial systems and the diffracted light only admitted into the objective, which can then be with an aperture of up to 1.4. Central dark field can also be used when the direct light is occluded in the objective itself and the diffracted light gathered. The limit for this technique comes when the light enters the specimen horizontally and then only diffracted light enters the objective. This method much advocated by Siedentopf [4] can reveal any particle however small provided it has a different refractive index than the medium it is in. Variations of this methodology can be introduced by allowing some direct light or by using annular illumination of varying aperture as through the Heine condenser made for many years in Leitz.
4.3 Phase Contrast Techniques Zernike made a significant discovery in making it possible without staining to make a refractive index change visible under the microscope under one condition [5,6] which is very important. This condition is that the refractive index varies rapidly between the object being examined in the specimen and its surrounds; ideally a sharp boundary is the most desired. This method was
Phase Imaging in Plant Cells and Tissues 55 to make predominantly a ring illuminator in the condenser and then introduce a ring in the objective with a cavity or increased height introducing thus a phase delay or advances in the objective to the light entering it and also causing the phase delayed light or accelerated light wave to have a different absorption in this region. The theory is shown graphically in Figure 4.1. Images that are produced using such methodology are shown in Figure 4.2. Note the halo around each boundary. There is also a further anomaly in the image. The outer part of the image has different contrast than the regions in the center and there is a further problem in
Phase plate
Phase change by preparation
Unaltered phase
Preparation
Light source
Figure 4.1 Formation of phase contrast image.
56
Chapter 4
10 µm
Figure 4.2 Inner epidermal nucleus from Allium cepa bulb scale.
that the image is only ideal for one wavelength and is suitable only for thin objects. The annular illumination introduces a large depth of focus to the images which can be problematic in thick specimens. This methodology was discovered by Zernike in 1930s [5 9]. It is much used in optical microscopy and also it has had applications in X-ray and electron microscopy. The method basically relies on illuminating the specimen with an annular beam of light although earlier methods used axial illumination and also a double annular illumination as in the interphako Zeiss microscope now obsolete. The usual method is a dark contrast system where the specimen has a darker image than the surrounds. The method works best for thin specimens and those of small extent. The main disadvantages of this approach are related to the artifacts that are caused by decay of contrast from the edges and a halo caused by absorption of light in this method [1]. The depth of focus is greater than in classical microscopy and results in disturbing fringes causing problems when imaging thick specimens. A new variation of through-focus imaging introduced by Nugent and colleagues and also by PhaseView in France works well for small angles and works excellently for specimens where the aperture of illumination is low [10]. The images are good for high apertures too but the accuracy declines dramatically with increased aperture. It is manufactured by Ultima in Australia and PhaseView in France. The phase ring used in classical phase microscopy is usually designed for a single wavelength but there have been versions which were achromatic and even apochromatic corrected for three colors.
Phase Imaging in Plant Cells and Tissues 57 (A)
(B)
90 µm
(C)
90 µm
90 µm
Figure 4.3 Onion inner epidermis from bulb scale in brightfield (A), low pass filter (B), and high pass filter (C).
Mitigation of the halos surrounding the phase delays are currently utilized by Nikon as apodized phase microscopy and reduce the halos considerably. Let us consider a specimen to be imaged by annular illumination with a phase ring in the objective which matches it. Figure 4.2 is of an onion inner epidermis nucleus showing contrast variations in black and white caused by phase differences due to different materials and different concentrations. The alternative method of phase contrast imaging uses the transport of energy formulation. Results are published by Barone-Nugent et al. [11] for fossils with relatively opaque specimens, cells with absorbing structures such as algae like Spirogyra and for phase view which has a similar development of algorithms was also done in a French company (Figure 4.3).
4.4 Becke Line in Optical Microscopy The method developed by Becke gives an easy determination of the refractive index of the desired object [12]. This is often used in mineral microscopy for looking at objects with a refractive index which one wishes to estimate and is applicable to botanical specimens. It is one of the earliest contrasting methods made between specimens and their surroundings and is due to their phase differences. Figure 4.4A explains how the Becke lines appear between transparent materials of different refractive indices. This figure demonstrates the case where a specimen that is denser than its surroundings (i.e., higher refractive index) is placed on the stage of the microscope. When elevating the objective or lowering the stage of the microscope, the Becke line will
58
Chapter 4 (A)
(B)
Becke line
Above focus Becke line inside Specimen Below focus Becke line outside
100 µm
Figure 4.4 (A) Becke line principle (top) and its formation at the inside of specimen (bottom). (B) Image of Becke lines in pear stone cells.
be formed inside the specimen at the above focus point. The formation of the Becke lines inside the specimen is illustrated in Figure 4.4A. A microscopical image that shows the formation of Becke lines inside the pear stone cells is shown in Figure 4.4B. The stone cells are surrounded by the line in black around each cell. This causes refractive index of cellulose encrusted with lignin and the depth of each stone cell creating a large phase difference between the cell and its surroundings. The surrounding medium is water which has a lower refractive index.
4.5 DIC Microscopy Since the transparent specimen is not able to be visualized by the human eye, the contrasting method plays an important role to overcome this problem. Of current microscopical methodologies for optical microscopy, the DIC is the most widely used and has fewest artifacts in imaging thick and absorbing specimens [13]. This kind of microscope works by illuminating the transparent specimen with dual polarized light sources that are split up from a single polarized light source via a Nomarski prism, such that they become slightly incoherent (i.e., the offset is illustrated by the red and blue dash lines that indicate the wavefront of each polarized light source) and perpendicular to each other. The wavelength of the incident light changes as it passes through the transparent specimen due to the refraction effect (usually becomes shorter due to the denser medium of the transparent specimen), hence resulting in the changes of phase. While still not able to be
Phase Imaging in Plant Cells and Tissues 59
Dark
DIC image
Light Polarizer
Nomarski prism
Interference fringes Objective Transparent specimen Stage Substage condenser Interference fringes
Nomarski prism Direction of propagation Polarizer From light source
Figure 4.5 DIC light microscopy.
visualized by the human eye, the image of the transparent specimen can be produced using the phase information of two polarizations. The separation between the two beams is less than the resolution of the object. By introducing the interference through the recombination of these dual polarized lights using the second Nomarski prism, the relative phase difference will be formed, hence enabling visualization for the detector. Figure 4.5 illustrates the process of how the image of transparent specimen can be produced.
60
Chapter 4
10 µm
Figure 4.6 An illustration of the process of image production in a DIC microscope.
Nomarski in 1955 [14] developed this methodology using polarized light but versions using unpolarized light also existed in the Interphako microscope. However, the highest apertures and the best focal depths were given by Nomarski’s methodology and it can also be extended to imaging in three dimensions by transfer of specimens in the visual field from one end to the other of the field of view thus creating two distinct illumination angles yielding a combined 3D effect as in Figure 4.6 (Sarafis, 1984 with the agreement also of Nomarski) [15]. It is possible to make a lower resolution image by DIC from data such as shown in the bright-field image in (Figure 4.7) on phase contrast imaging using software but they are not as good as direct DIC microscopy.
4.6 Hoffmann Modulation Contrast HMC is a method that makes phase gradients to be displayed at different levels of intensity relative to the background, such that the transparent object can be visualized on a microscope [1]. It can be retrofitted to any microscope and gives relief contrast, high resolving power in the images, and a small depth of focus [16]. The HMC microscope works by placing a modulator at the back focal plane of the objective. This modulator consists of three regions that have different transmission levels (usually 1%, 15%, and 100%). Also, a slit has to be placed at the front focal plane of the substage condenser and calibrated to allow the incident light passing through the region of the modulator that has 15% transmission level, such that the background of the image
Phase Imaging in Plant Cells and Tissues 61
90 µm
Figure 4.7 DIC image produced by software from data shown in Figure 4.3.
becomes uniformly gray. Upon placing the specimen on the stage of HMC microscope, some of the incident light will be refracted and passed through either the 100% or the 1% region of the modulator. Hence, the image of the specimen, as visualized by the slit, will appear to be bright or dark, respectively. The incident light that is not refracted will still be passing through the 15% region of the modulator. While the HMC microscope functions like a DIC, it is immune to birefringence that can be caused by some specimens. Note that the specimen must be rotated, allowing the asymmetries and symmetries in the specimen to be differentiated. The structure of an HMC microscope and its operating concept is shown in Figure 4.8. It seems that this method has now lapsed as a patent and is still marketed under names such as Varel by Zeiss [17] and by Nikon as NAMC [18]. The author is unaware of its use for plant cells and tissue imaging thus far but it does have strong potential in his field of endeavor.
4.7 Adaptive Optics Microscopy Derived from astronomy primarily, it has so far only been used for fluorescence although it should find use also in transmission and reflection imaging with quasicoherent light. Recently, Zeiss has had precommercial instruments offering this technology. A typical example of applying adaptive optics in the area of microscopy is demonstrated by Poland et al. [19]. The experiment of this demonstration utilized a laser-scanning confocal microscope (later reconfigured as a multiphoton fluorescent microscope) with DMM as its
62
Chapter 4 Dark image
Gray image
Bright image
Modulator 1%
15%
100%
Objective
Specimen
Substage condenser
Slit Polarizer P2 Polarizer P1
Light source
Figure 4.8 HMC microscope and its operating concept.
adaptive optics element. The optimization of the image quality of the sample is based on the shape calibration of DMM using an algorithm to mitigate aberration issue. This experiment produced results that illustrated the capability of single aberration correction in enhancing the image quality over the conventional wide-field fluorescence microscopy. Figure 4.9 shows an example of using adaptive optics with a single plane illumination microscope on Arabidopsis root for a fluorescent case from work led by John Girkin of Durham University. Such microscopy method is identical to the one performed in Ref. [20].
4.8 Interferometric Microscopy The principle of the SLIM in conjunction with a Zeiss manufactured “Axio Observer Z1” phase contrast microscope is shown in Figure 4.10 while imaging neurons grown in vitro (taken from Ref. [21]). A halogen lamp is used as the light source of the microscope. The specimen on the stage can be pinpointed by the incident lights with the aid of the
Phase Imaging in Plant Cells and Tissues 63 (A)
(B)
175 µm
175 µm
Figure 4.9 Adaptive optics in Arabidopsis root sections (A) as produced from fluorescence microscope (B) after image processing.
Halogen lamp filament
Collector lens
Field diaphragm Aperture
SLIM module Condenser annulus
Condenser lens
0
CCD Specimen
Objective
Backfocal plane
Fourier lens L2
Image plane
π
3π/2
Phase rings
LCPM
Tube lens
Mirror
π/2
Fourier lens L1
Beam splitter
Figure 4.10 Configuration of SLIM microscope (left). The images of variation of phase rings as captured by the CCD (right).
64
Chapter 4 106 79 53 26 0
Figure 4.11 Image acquired by SLIM from the outer onion epidermis. Source: Courtesy of Mustafa Mir and Gabriel Popescu, Quantitative Light Imaging Laboratory, University of Illinois at Urbana-Champaign.
annulus condenser of the microscope. The phase ring that is fitted on the objective of the conventional phase contrast microscope works as a phase-shifter and attenuator. The postprocessing procedure can be performed by the SLIM module on the image of the specimen, which is redirected from the microscope to the image plane via a tube lens. The Fourier lens L1 serves as a relay between the back focal plane of the objective and the surface of the liquid crystal phase modulator (LCPM). The phase delay can be precisely modulated between the scattered and the unscattered components to visualize the variation of masks on the LCPM. Consequently, the image of the specimen will be captured by the CCD produced by the Fourier lens L2 in the SLIM module. Unlike, in the DIC microscopy of Nomarski double beam microscopy with complete separation of the two partially coherent beams was in vogue from many commercial sources. Polskie Zakłady Optyczne (PZO) had the Pluta Interference microscope, Leitz had a double microscope for this purpose, Zeiss from West Germany had a Jamin Lebedeff interference microscope, and Zeiss Jena had the Interphako. None of these are any longer in production. Recently, Popescu from Illinois University has developed a commercial microscope called SLIM. Figure 4.11 shows the outer epidermis of an onion scale. Image of a cell layer from white onion skin was acquired using SLIM. The image covers an area of 1939 3 1453 µm2 created by stitching 5 3 5 images together. At each of the 25 locations, a total of 34 z-sections were acquired spaced 4.5 µm apart and the maximum value from each slice was projected to create the image shown. The inset shows a
Phase Imaging in Plant Cells and Tissues 65 250 3 400 µm2 detail of an area from this image. The yellow scale bar is 250 µm and the color bar shows optical path length in nanometers. SLIM microscopy has been extended recently to super resolving microscopy by [22].
4.9 Second Harmonic Imaging Microscopy SHIM was originated with confocal microscopy in transmission. Double photon absorption by orientated structures such as cell walls, starch grains, and chloroplasts is remitted in a shorter wavelength. The fluorescence that can result from this is always a longer wavelength than the remitted light which is at a shorter wavelength than the irradiation with pulsed wavelength. This kind of microscope obtains the half wavelength of the light source using the nonlinear optical effect (i.e., second-harmonic generation) caused by the noncentrosymmetric structure of the specimen as a contrasting method to the image of the specimen. Such method is equivalent to the way that a conventional optical microscope obtaining contrast of specimen by detecting variations in optical density, path length, and/or the refractive index of the specimen. Campagnola and Leow [22] specify the advantages of this microscopy as unsusceptible to phototoxicity or photo-bleaching and also molecules with exogenous probes not necessarily to be labeled since many of the biological structures can produce strong second-harmonic generation signals, and hence will not affect the functioning of these biological systems. This is commercially available from Till photonics and Leica. In the latter, only the light emitted forward is imaged, but in the Till microscope one collects both directions and these give different information. A conventional laser-scanning double-photon microscope can also be simply upgraded to achieve the SHIM’s capability with minor modification [23]. The paper by Reshak et al. [24] shows the case in chloroplasts of a moss leaf.
4.10 Magnetic Resonance Imaging of Plants Showing the Flow Component After early work with Sir Paul Callaghan and Xia, MRI was used by Sarafis and Campbell (25) to demonstrate flow by imaging the xylem and phloem of plants. Figures 4.12 and 4.13 show the power of this technology which examines the flow of the fluids in these channels and is unlike Doppler methods in optical microscopy [26]. The Doppler methodologies are not commercial whereas the MRI is done on commercial machines which are not commercial; these latter depend on the carrying of particulates in the flow streams, but the MRI imaging is utterly independent of these components.
66
Chapter 4
Field of view (FOV): 12 × 12 mm Matrix size: 128 × 128 Rapid acquisition with refocused echoes (RARE) factors: 8 Repetition time (TR): 1250 ms Echo time (TE): 5 ms. 125
Amplitude (water density or content) max = 125 (arbitrary units)
0
10
0.5
Phase shift (velocity) mm/s
Phase shift (velocity) mm/s
0.0
0.0
Figure 4.12 The cross-section of Cucurbita by MRI from intact plant showing distribution of water and flow in the xylem (left) and phloem (right).
Field of view (FOV): 12.5 × 12.5 nm Diameter: 7.6 mm Stationary water/pixel average linear velocity/pixel (%) (mm/s) (A)
(B)
0.4
0 0
100%
0
2
Figure 4.13 Poplar stem sections imaged by MRI showing water distribution (A) and flow in the xylem and phloem (B).
Phase Imaging in Plant Cells and Tissues 67 Newer emerging methods involving the hyperpolarization of water will change the imaging further and make it less demanding in time and increase the signal-to-noise component considerably by at least one and probably more orders of magnitude. Windt et al. [26,27] originally at Wageningen University Center for MRI showed an example of flow imaging in plants in Figures 4.12 and 4.13. MRI microscopy can also be used to show different phases such as water, oil, and essential components [28,29].
Acknowledgments Grateful thanks to: Brendan Allman for providing the phase images (Figures 4.3 and 4.7), Melbourne University. John Girkin for adaptive optics (Figure 4.9), Durham University. Gabriel Popescu for interference image by SLIM (Figures 4.1 and 4.11), University of Illionis. Henk Van As for providing MRI images (Figures 4.12 and 4.13), University of Wageningen. Rabia Ghaffar for acquiring phase contrast, DIC, and bright-field images (Figures 4.1, 4.2, 4.4, and 4.6) and in compiling the article, University of Vienna. Chow Yii Pui for assistance in the final compilation of this book chapter and the images (Figures 4.5, 4.8, and 4.10), University of Adelaide. With sincere regrets we inform that Prof. Vassilious Sarafis has passed away just before his chapter was finalized for publication. On the behalf of the editors we express our condolences to his family and dedicate his chapter to a memory of an extraordinary and multidisciplinary scientist, a colorful and unique person and a dear colleague.
References [1] R. Wayne, Light and Video Microscopy, Elsevier, New York, NY, 2009, pp. 80 81 [2] S.H. Gage, Modern dark-field microscopy and the history of its development, Am. Microsc. Soc. (39) (1920) 95 141. [3] S.H. Gage, The Microscope. Dark-Field Edition, Comstock Publishing Co., Ithaca, NY, 1925. [4] H. Siedentopf, On the rendering visible of ultra-microscopic particles and of ultra-microscopic bacteria, J. R. Microsc. Soc. 23(573 578) (1903) 5. [5] F. Zernike, Phase contrast, a new method of microscopic observation of transparent objects, Physica II(9) (1942) 947 986. [6] F. Zernike, Phase-contrast, a new method for microscopic observation of transparent objects, Physica I(9) (1942) 686 698. [7] F. Zernike, Phase-contrast, a new method for microscopic observation of transparent object, in: A. Bouwers (Ed.), Achievements in Optics, Elsevier, New York, NY, 1946, pp. 116 135. [8] F. Zernike, Observing the phase of light waves, Science 107 (1948) 463. [9] G.L.E. Turner, Frits Zernike, 1888 1966, R. Microsc. Soc. 17 (1982) 100 101. [10] D. Paganin, K.A. Nugent, Noninterferometric phase imaging with partially coherent light, Phys. Rev. Lett. 80(12) (1998) 2586 2589.
68
Chapter 4
[11] E.D. Barone-Nugent, A. Barty, K.A. Nugent, Quantitative phase-amplitude microscopy I: optical microscopy, J. Microsc. 206(3) (2002) 194 203. [12] A. Allaby, M. Allaby, Becke Line Test, A Dictionary of Earth Sciences, January 1999 (Online). Available from: ,http://www.encyclopedia.com/doc/1O13-Beckelinetest.html/ . (accessed 16.03.2012). [13] G. Nomarski, Interferential polarizing device for study of phase objects, US Patent 2,924,142, 9 February 1960. [14] G. Nomarski, Microinterfe´rome`tre diffe´rentiel a` ondes polarise´es, J. Phys. Radium, Paris 16 (1955) 9S 11S. [15] V. Sarafis, X-ray microscopy as a possible tool for the investigation of plant cells, Springer Series in Optical Sciences, X-ray microscopy, 1984. [16] Modulation Optics, Making Transparent Specimens Clear and Vividly Detailed, Modulation Optics, 2012 (Online). Available from: ,http://www.modulationoptics.com/. (accessed 16.03.2012). [17] B. Hohman, E. Keller, Varel: A New Contrasting Method for Microscopy, Carl Zeiss Inc., Thornwood, NY, 2001. [18] Laboratorytalk, NAMC Enhances Image Sharpness and Definition, Laboratorytalk, 9 September 2009 (Online). Available from: ,http://www.laboratorytalk.com/news/nik/nik223.html/ . (accessed 16.03.2012). [19] S.P. Poland, A.J. Wright, S. Cobb, J.C. Vijverberg, J.M. Girkin, A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes, Micron 42(4) (2011) 318 323. [20] J.M. Taylor, C.D. Saunter, G.D. Love, J.M. Girkin, Heart synchronization for SPIM microscopy of living zebra fish, in: SPIE 7904, San Francisco, 2011. [21] M. Mir, Z. Wanga, Z. Shen, M. Bednarzd, R. Bashira, I. Golding, et al., Optical measurement of cycle dependent cell growth, Proc. Natl. Acad. Sci. U.S.A. 108(32) (2011) 1 6. [22] S.D. Babacan, Z. Wanga, M. Do, G. Popescu, Cell imaging beyond the diffraction limit using sparse deconvolution spatial light interference microscopy, Biomed. Opt. Express 2(7) (2011) 1815 1857. [23] P.J. Campagnola, L.M. Loew, Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms, Nat. Biotechnol. 21(11) (2003) 1356 1360. [24] A.H. Reshak, V. Sarafis, R. Heintzmann, Second harmonic imaging of chloroplasts using the two-photon laser scanning microscope, Micron 40(3) (2009) 378 385. [25] Y. Xia, V. Sarafis, E.O. Campbell, P.T. Callaghan, Noninvasive imaging of water flow in plants by NMR microscopy, Protoplasma 173(3 4) (1993) 170 176. [26] C.W. Windt, Nuclear Magnetic Resonance Imaging of Sap Flow in Plants, University of Wageningen, Wageningen, 2007. [27] C.W. Windt, F.J. Vergeldt, P.A. DeJager, H.V. As, MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco, Plant Cell Environ. 29(9) (2006) 1715 1729. [28] V. Sarafis, H. Rumel, J. Pope, W. Kuhn, Non-invasive histochemistry of plant materials by magnetic resonance microscopy, Protoplasma 159(1) (1990) 70 73. [29] J. Pope, V. Sarafis, NMR microscopy, Chem. Aust. 57(7) (1990) 221 224.
Phase Imaging in Plant Cells and Tissues Vassilios Sarafis Botany School University of Melbourne, Vic, Australia Mathematics and Physics School, University of Queensland, Brisbane St Lucia, QLD, Australia Biomedical Science and Engineering School, University of Adelaide, SA, Australia Birla Institute of Technology and Science, Hyderabad, Andhra Pradesh, India
Editor: Zeev Zalevsky
Phase imaging in plant cells and tissues depends on the boundaries between places where concentration of water is different or there is a boundary between water and oil containing compartments. This chapter has listed 10 microscopy methods to make the boundaries to be visible to the human eye so that they can be well studied. These methods can be applied for transparent specimens of different characteristics. The out of focus imaging is the first and the earliest method that produces contrast at the boundary of the specimen using the out of focus approach. The dark field microscopy creates dark background around the specimen using the elimination of the unscattered beam from the image of the specimen. The phase contrast techniques, presented Chapter 1, allow a living specimen to be studied due to its exclusion of staining showing the boundaries where refractive index changes. There are also other types of contrast microscopy. One is to use Becke line test to create contrast between the boundaries of specimens. Differential contrast microscopy (DIC), presented in Chapter 2, is another type of contrast microscopy. It uses the interference of dual polarizations to form visible images of specimens due to the phase difference. Also, the Hoffman modulation contrast (HMC) microscope is able to vary the contrast of different regions within a specimen using its phase gradients. The adaptive optics microscopy, on the other hand, uses adaptive optics elements such as deformable membrane mirror (DMM) or spatial light modulators to optimize the image quality of the specimen, usually in fluorescence. The interferometric microscopy, also uses phase contrast technique with the addition of a spatial light interference microscopy (SLIM) module to capture the image of a specimen. Biomedical Optical Phase Microscopy and Nanoscopy © 2013 Elsevier Inc. All rights reserved.
53
54
Chapter 4
The second harmonic imaging microscopy (SHIM) obtains the half wavelength signal which is due to the nonlinear optical effect of the specimen for imaging. The final part of this chapter shows the flowing image in the plants using the magnetic resonance imaging (MRI) as well as static water and lipids.
4.1 Out of Focus Imaging The earliest method for observing phase variations is imaging at various focal positions. This earliest method is known as out of focus contrast. Let us consider an object in focus. It is utterly transparent but has a boundary separating it from water with the same refractive indices in and out of the boundary. If in true focus we shall see nothing in a transparent unstained specimen. Just out of focus it will have contrast which reverses as we go through true focus to the other side off the true focus. The contrast is due to partial coherence of the light waves originating from parts of the object prior to and after the object being observed [1]. Using Adobe Photoshop, it is possible to increase the contrast of such images considerably. Such a method has been used by some Indian botanist scientists to observe enhanced contrast in pollen grains mounted in water. In that case, there is a difference in concentration of materials in the pollen grain and the outside which is pure water and this enhances the contrast due to refraction at the boundaries as well.
4.2 Dark Field An excellent method for visualizing boundaries is dark field. In this method, direct light from the microscope condenser is excluded from entering the objective [2,3]. A high aperture may be used for illumination up to 1.42 in commercial systems and the diffracted light only admitted into the objective, which can then be with an aperture of up to 1.4. Central dark field can also be used when the direct light is occluded in the objective itself and the diffracted light gathered. The limit for this technique comes when the light enters the specimen horizontally and then only diffracted light enters the objective. This method much advocated by Siedentopf [4] can reveal any particle however small provided it has a different refractive index than the medium it is in. Variations of this methodology can be introduced by allowing some direct light or by using annular illumination of varying aperture as through the Heine condenser made for many years in Leitz.
4.3 Phase Contrast Techniques Zernike made a significant discovery in making it possible without staining to make a refractive index change visible under the microscope under one condition [5,6] which is very important. This condition is that the refractive index varies rapidly between the object being examined in the specimen and its surrounds; ideally a sharp boundary is the most desired. This method was
Phase Imaging in Plant Cells and Tissues 55 to make predominantly a ring illuminator in the condenser and then introduce a ring in the objective with a cavity or increased height introducing thus a phase delay or advances in the objective to the light entering it and also causing the phase delayed light or accelerated light wave to have a different absorption in this region. The theory is shown graphically in Figure 4.1. Images that are produced using such methodology are shown in Figure 4.2. Note the halo around each boundary. There is also a further anomaly in the image. The outer part of the image has different contrast than the regions in the center and there is a further problem in
Phase plate
Phase change by preparation
Unaltered phase
Preparation
Light source
Figure 4.1 Formation of phase contrast image.
56
Chapter 4
10 µm
Figure 4.2 Inner epidermal nucleus from Allium cepa bulb scale.
that the image is only ideal for one wavelength and is suitable only for thin objects. The annular illumination introduces a large depth of focus to the images which can be problematic in thick specimens. This methodology was discovered by Zernike in 1930s [5 9]. It is much used in optical microscopy and also it has had applications in X-ray and electron microscopy. The method basically relies on illuminating the specimen with an annular beam of light although earlier methods used axial illumination and also a double annular illumination as in the interphako Zeiss microscope now obsolete. The usual method is a dark contrast system where the specimen has a darker image than the surrounds. The method works best for thin specimens and those of small extent. The main disadvantages of this approach are related to the artifacts that are caused by decay of contrast from the edges and a halo caused by absorption of light in this method [1]. The depth of focus is greater than in classical microscopy and results in disturbing fringes causing problems when imaging thick specimens. A new variation of through-focus imaging introduced by Nugent and colleagues and also by PhaseView in France works well for small angles and works excellently for specimens where the aperture of illumination is low [10]. The images are good for high apertures too but the accuracy declines dramatically with increased aperture. It is manufactured by Ultima in Australia and PhaseView in France. The phase ring used in classical phase microscopy is usually designed for a single wavelength but there have been versions which were achromatic and even apochromatic corrected for three colors.
Phase Imaging in Plant Cells and Tissues 57 (A)
(B)
90 µm
(C)
90 µm
90 µm
Figure 4.3 Onion inner epidermis from bulb scale in brightfield (A), low pass filter (B), and high pass filter (C).
Mitigation of the halos surrounding the phase delays are currently utilized by Nikon as apodized phase microscopy and reduce the halos considerably. Let us consider a specimen to be imaged by annular illumination with a phase ring in the objective which matches it. Figure 4.2 is of an onion inner epidermis nucleus showing contrast variations in black and white caused by phase differences due to different materials and different concentrations. The alternative method of phase contrast imaging uses the transport of energy formulation. Results are published by Barone-Nugent et al. [11] for fossils with relatively opaque specimens, cells with absorbing structures such as algae like Spirogyra and for phase view which has a similar development of algorithms was also done in a French company (Figure 4.3).
4.4 Becke Line in Optical Microscopy The method developed by Becke gives an easy determination of the refractive index of the desired object [12]. This is often used in mineral microscopy for looking at objects with a refractive index which one wishes to estimate and is applicable to botanical specimens. It is one of the earliest contrasting methods made between specimens and their surroundings and is due to their phase differences. Figure 4.4A explains how the Becke lines appear between transparent materials of different refractive indices. This figure demonstrates the case where a specimen that is denser than its surroundings (i.e., higher refractive index) is placed on the stage of the microscope. When elevating the objective or lowering the stage of the microscope, the Becke line will
58
Chapter 4 (A)
(B)
Becke line
Above focus Becke line inside Specimen Below focus Becke line outside
100 µm
Figure 4.4 (A) Becke line principle (top) and its formation at the inside of specimen (bottom). (B) Image of Becke lines in pear stone cells.
be formed inside the specimen at the above focus point. The formation of the Becke lines inside the specimen is illustrated in Figure 4.4A. A microscopical image that shows the formation of Becke lines inside the pear stone cells is shown in Figure 4.4B. The stone cells are surrounded by the line in black around each cell. This causes refractive index of cellulose encrusted with lignin and the depth of each stone cell creating a large phase difference between the cell and its surroundings. The surrounding medium is water which has a lower refractive index.
4.5 DIC Microscopy Since the transparent specimen is not able to be visualized by the human eye, the contrasting method plays an important role to overcome this problem. Of current microscopical methodologies for optical microscopy, the DIC is the most widely used and has fewest artifacts in imaging thick and absorbing specimens [13]. This kind of microscope works by illuminating the transparent specimen with dual polarized light sources that are split up from a single polarized light source via a Nomarski prism, such that they become slightly incoherent (i.e., the offset is illustrated by the red and blue dash lines that indicate the wavefront of each polarized light source) and perpendicular to each other. The wavelength of the incident light changes as it passes through the transparent specimen due to the refraction effect (usually becomes shorter due to the denser medium of the transparent specimen), hence resulting in the changes of phase. While still not able to be
Phase Imaging in Plant Cells and Tissues 59
Dark
DIC image
Light Polarizer
Nomarski prism
Interference fringes Objective Transparent specimen Stage Substage condenser Interference fringes
Nomarski prism Direction of propagation Polarizer From light source
Figure 4.5 DIC light microscopy.
visualized by the human eye, the image of the transparent specimen can be produced using the phase information of two polarizations. The separation between the two beams is less than the resolution of the object. By introducing the interference through the recombination of these dual polarized lights using the second Nomarski prism, the relative phase difference will be formed, hence enabling visualization for the detector. Figure 4.5 illustrates the process of how the image of transparent specimen can be produced.
60
Chapter 4
10 µm
Figure 4.6 An illustration of the process of image production in a DIC microscope.
Nomarski in 1955 [14] developed this methodology using polarized light but versions using unpolarized light also existed in the Interphako microscope. However, the highest apertures and the best focal depths were given by Nomarski’s methodology and it can also be extended to imaging in three dimensions by transfer of specimens in the visual field from one end to the other of the field of view thus creating two distinct illumination angles yielding a combined 3D effect as in Figure 4.6 (Sarafis, 1984 with the agreement also of Nomarski) [15]. It is possible to make a lower resolution image by DIC from data such as shown in the bright-field image in (Figure 4.7) on phase contrast imaging using software but they are not as good as direct DIC microscopy.
4.6 Hoffmann Modulation Contrast HMC is a method that makes phase gradients to be displayed at different levels of intensity relative to the background, such that the transparent object can be visualized on a microscope [1]. It can be retrofitted to any microscope and gives relief contrast, high resolving power in the images, and a small depth of focus [16]. The HMC microscope works by placing a modulator at the back focal plane of the objective. This modulator consists of three regions that have different transmission levels (usually 1%, 15%, and 100%). Also, a slit has to be placed at the front focal plane of the substage condenser and calibrated to allow the incident light passing through the region of the modulator that has 15% transmission level, such that the background of the image
Phase Imaging in Plant Cells and Tissues 61
90 µm
Figure 4.7 DIC image produced by software from data shown in Figure 4.3.
becomes uniformly gray. Upon placing the specimen on the stage of HMC microscope, some of the incident light will be refracted and passed through either the 100% or the 1% region of the modulator. Hence, the image of the specimen, as visualized by the slit, will appear to be bright or dark, respectively. The incident light that is not refracted will still be passing through the 15% region of the modulator. While the HMC microscope functions like a DIC, it is immune to birefringence that can be caused by some specimens. Note that the specimen must be rotated, allowing the asymmetries and symmetries in the specimen to be differentiated. The structure of an HMC microscope and its operating concept is shown in Figure 4.8. It seems that this method has now lapsed as a patent and is still marketed under names such as Varel by Zeiss [17] and by Nikon as NAMC [18]. The author is unaware of its use for plant cells and tissue imaging thus far but it does have strong potential in his field of endeavor.
4.7 Adaptive Optics Microscopy Derived from astronomy primarily, it has so far only been used for fluorescence although it should find use also in transmission and reflection imaging with quasicoherent light. Recently, Zeiss has had precommercial instruments offering this technology. A typical example of applying adaptive optics in the area of microscopy is demonstrated by Poland et al. [19]. The experiment of this demonstration utilized a laser-scanning confocal microscope (later reconfigured as a multiphoton fluorescent microscope) with DMM as its
62
Chapter 4 Dark image
Gray image
Bright image
Modulator 1%
15%
100%
Objective
Specimen
Substage condenser
Slit Polarizer P2 Polarizer P1
Light source
Figure 4.8 HMC microscope and its operating concept.
adaptive optics element. The optimization of the image quality of the sample is based on the shape calibration of DMM using an algorithm to mitigate aberration issue. This experiment produced results that illustrated the capability of single aberration correction in enhancing the image quality over the conventional wide-field fluorescence microscopy. Figure 4.9 shows an example of using adaptive optics with a single plane illumination microscope on Arabidopsis root for a fluorescent case from work led by John Girkin of Durham University. Such microscopy method is identical to the one performed in Ref. [20].
4.8 Interferometric Microscopy The principle of the SLIM in conjunction with a Zeiss manufactured “Axio Observer Z1” phase contrast microscope is shown in Figure 4.10 while imaging neurons grown in vitro (taken from Ref. [21]). A halogen lamp is used as the light source of the microscope. The specimen on the stage can be pinpointed by the incident lights with the aid of the
Phase Imaging in Plant Cells and Tissues 63 (A)
(B)
175 µm
175 µm
Figure 4.9 Adaptive optics in Arabidopsis root sections (A) as produced from fluorescence microscope (B) after image processing.
Halogen lamp filament
Collector lens
Field diaphragm Aperture
SLIM module Condenser annulus
Condenser lens
0
CCD Specimen
Objective
Backfocal plane
Fourier lens L2
Image plane
π
3π/2
Phase rings
LCPM
Tube lens
Mirror
π/2
Fourier lens L1
Beam splitter
Figure 4.10 Configuration of SLIM microscope (left). The images of variation of phase rings as captured by the CCD (right).
64
Chapter 4 106 79 53 26 0
Figure 4.11 Image acquired by SLIM from the outer onion epidermis. Source: Courtesy of Mustafa Mir and Gabriel Popescu, Quantitative Light Imaging Laboratory, University of Illinois at Urbana-Champaign.
annulus condenser of the microscope. The phase ring that is fitted on the objective of the conventional phase contrast microscope works as a phase-shifter and attenuator. The postprocessing procedure can be performed by the SLIM module on the image of the specimen, which is redirected from the microscope to the image plane via a tube lens. The Fourier lens L1 serves as a relay between the back focal plane of the objective and the surface of the liquid crystal phase modulator (LCPM). The phase delay can be precisely modulated between the scattered and the unscattered components to visualize the variation of masks on the LCPM. Consequently, the image of the specimen will be captured by the CCD produced by the Fourier lens L2 in the SLIM module. Unlike, in the DIC microscopy of Nomarski double beam microscopy with complete separation of the two partially coherent beams was in vogue from many commercial sources. Polskie Zakłady Optyczne (PZO) had the Pluta Interference microscope, Leitz had a double microscope for this purpose, Zeiss from West Germany had a Jamin Lebedeff interference microscope, and Zeiss Jena had the Interphako. None of these are any longer in production. Recently, Popescu from Illinois University has developed a commercial microscope called SLIM. Figure 4.11 shows the outer epidermis of an onion scale. Image of a cell layer from white onion skin was acquired using SLIM. The image covers an area of 1939 3 1453 µm2 created by stitching 5 3 5 images together. At each of the 25 locations, a total of 34 z-sections were acquired spaced 4.5 µm apart and the maximum value from each slice was projected to create the image shown. The inset shows a
Phase Imaging in Plant Cells and Tissues 65 250 3 400 µm2 detail of an area from this image. The yellow scale bar is 250 µm and the color bar shows optical path length in nanometers. SLIM microscopy has been extended recently to super resolving microscopy by [22].
4.9 Second Harmonic Imaging Microscopy SHIM was originated with confocal microscopy in transmission. Double photon absorption by orientated structures such as cell walls, starch grains, and chloroplasts is remitted in a shorter wavelength. The fluorescence that can result from this is always a longer wavelength than the remitted light which is at a shorter wavelength than the irradiation with pulsed wavelength. This kind of microscope obtains the half wavelength of the light source using the nonlinear optical effect (i.e., second-harmonic generation) caused by the noncentrosymmetric structure of the specimen as a contrasting method to the image of the specimen. Such method is equivalent to the way that a conventional optical microscope obtaining contrast of specimen by detecting variations in optical density, path length, and/or the refractive index of the specimen. Campagnola and Leow [22] specify the advantages of this microscopy as unsusceptible to phototoxicity or photo-bleaching and also molecules with exogenous probes not necessarily to be labeled since many of the biological structures can produce strong second-harmonic generation signals, and hence will not affect the functioning of these biological systems. This is commercially available from Till photonics and Leica. In the latter, only the light emitted forward is imaged, but in the Till microscope one collects both directions and these give different information. A conventional laser-scanning double-photon microscope can also be simply upgraded to achieve the SHIM’s capability with minor modification [23]. The paper by Reshak et al. [24] shows the case in chloroplasts of a moss leaf.
4.10 Magnetic Resonance Imaging of Plants Showing the Flow Component After early work with Sir Paul Callaghan and Xia, MRI was used by Sarafis and Campbell (25) to demonstrate flow by imaging the xylem and phloem of plants. Figures 4.12 and 4.13 show the power of this technology which examines the flow of the fluids in these channels and is unlike Doppler methods in optical microscopy [26]. The Doppler methodologies are not commercial whereas the MRI is done on commercial machines which are not commercial; these latter depend on the carrying of particulates in the flow streams, but the MRI imaging is utterly independent of these components.
66
Chapter 4
Field of view (FOV): 12 × 12 mm Matrix size: 128 × 128 Rapid acquisition with refocused echoes (RARE) factors: 8 Repetition time (TR): 1250 ms Echo time (TE): 5 ms. 125
Amplitude (water density or content) max = 125 (arbitrary units)
0
10
0.5
Phase shift (velocity) mm/s
Phase shift (velocity) mm/s
0.0
0.0
Figure 4.12 The cross-section of Cucurbita by MRI from intact plant showing distribution of water and flow in the xylem (left) and phloem (right).
Field of view (FOV): 12.5 × 12.5 nm Diameter: 7.6 mm Stationary water/pixel average linear velocity/pixel (%) (mm/s) (A)
(B)
0.4
0 0
100%
0
2
Figure 4.13 Poplar stem sections imaged by MRI showing water distribution (A) and flow in the xylem and phloem (B).
Phase Imaging in Plant Cells and Tissues 67 Newer emerging methods involving the hyperpolarization of water will change the imaging further and make it less demanding in time and increase the signal-to-noise component considerably by at least one and probably more orders of magnitude. Windt et al. [26,27] originally at Wageningen University Center for MRI showed an example of flow imaging in plants in Figures 4.12 and 4.13. MRI microscopy can also be used to show different phases such as water, oil, and essential components [28,29].
Acknowledgments Grateful thanks to: Brendan Allman for providing the phase images (Figures 4.3 and 4.7), Melbourne University. John Girkin for adaptive optics (Figure 4.9), Durham University. Gabriel Popescu for interference image by SLIM (Figures 4.1 and 4.11), University of Illionis. Henk Van As for providing MRI images (Figures 4.12 and 4.13), University of Wageningen. Rabia Ghaffar for acquiring phase contrast, DIC, and bright-field images (Figures 4.1, 4.2, 4.4, and 4.6) and in compiling the article, University of Vienna. Chow Yii Pui for assistance in the final compilation of this book chapter and the images (Figures 4.5, 4.8, and 4.10), University of Adelaide. With sincere regrets we inform that Prof. Vassilious Sarafis has passed away just before his chapter was finalized for publication. On the behalf of the editors we express our condolences to his family and dedicate his chapter to a memory of an extraordinary and multidisciplinary scientist, a colorful and unique person and a dear colleague.
References [1] R. Wayne, Light and Video Microscopy, Elsevier, New York, NY, 2009, pp. 80 81 [2] S.H. Gage, Modern dark-field microscopy and the history of its development, Am. Microsc. Soc. (39) (1920) 95 141. [3] S.H. Gage, The Microscope. Dark-Field Edition, Comstock Publishing Co., Ithaca, NY, 1925. [4] H. Siedentopf, On the rendering visible of ultra-microscopic particles and of ultra-microscopic bacteria, J. R. Microsc. Soc. 23(573 578) (1903) 5. [5] F. Zernike, Phase contrast, a new method of microscopic observation of transparent objects, Physica II(9) (1942) 947 986. [6] F. Zernike, Phase-contrast, a new method for microscopic observation of transparent objects, Physica I(9) (1942) 686 698. [7] F. Zernike, Phase-contrast, a new method for microscopic observation of transparent object, in: A. Bouwers (Ed.), Achievements in Optics, Elsevier, New York, NY, 1946, pp. 116 135. [8] F. Zernike, Observing the phase of light waves, Science 107 (1948) 463. [9] G.L.E. Turner, Frits Zernike, 1888 1966, R. Microsc. Soc. 17 (1982) 100 101. [10] D. Paganin, K.A. Nugent, Noninterferometric phase imaging with partially coherent light, Phys. Rev. Lett. 80(12) (1998) 2586 2589.
68
Chapter 4
[11] E.D. Barone-Nugent, A. Barty, K.A. Nugent, Quantitative phase-amplitude microscopy I: optical microscopy, J. Microsc. 206(3) (2002) 194 203. [12] A. Allaby, M. Allaby, Becke Line Test, A Dictionary of Earth Sciences, January 1999 (Online). Available from: ,http://www.encyclopedia.com/doc/1O13-Beckelinetest.html/ . (accessed 16.03.2012). [13] G. Nomarski, Interferential polarizing device for study of phase objects, US Patent 2,924,142, 9 February 1960. [14] G. Nomarski, Microinterfe´rome`tre diffe´rentiel a` ondes polarise´es, J. Phys. Radium, Paris 16 (1955) 9S 11S. [15] V. Sarafis, X-ray microscopy as a possible tool for the investigation of plant cells, Springer Series in Optical Sciences, X-ray microscopy, 1984. [16] Modulation Optics, Making Transparent Specimens Clear and Vividly Detailed, Modulation Optics, 2012 (Online). Available from: ,http://www.modulationoptics.com/. (accessed 16.03.2012). [17] B. Hohman, E. Keller, Varel: A New Contrasting Method for Microscopy, Carl Zeiss Inc., Thornwood, NY, 2001. [18] Laboratorytalk, NAMC Enhances Image Sharpness and Definition, Laboratorytalk, 9 September 2009 (Online). Available from: ,http://www.laboratorytalk.com/news/nik/nik223.html/ . (accessed 16.03.2012). [19] S.P. Poland, A.J. Wright, S. Cobb, J.C. Vijverberg, J.M. Girkin, A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes, Micron 42(4) (2011) 318 323. [20] J.M. Taylor, C.D. Saunter, G.D. Love, J.M. Girkin, Heart synchronization for SPIM microscopy of living zebra fish, in: SPIE 7904, San Francisco, 2011. [21] M. Mir, Z. Wanga, Z. Shen, M. Bednarzd, R. Bashira, I. Golding, et al., Optical measurement of cycle dependent cell growth, Proc. Natl. Acad. Sci. U.S.A. 108(32) (2011) 1 6. [22] S.D. Babacan, Z. Wanga, M. Do, G. Popescu, Cell imaging beyond the diffraction limit using sparse deconvolution spatial light interference microscopy, Biomed. Opt. Express 2(7) (2011) 1815 1857. [23] P.J. Campagnola, L.M. Loew, Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms, Nat. Biotechnol. 21(11) (2003) 1356 1360. [24] A.H. Reshak, V. Sarafis, R. Heintzmann, Second harmonic imaging of chloroplasts using the two-photon laser scanning microscope, Micron 40(3) (2009) 378 385. [25] Y. Xia, V. Sarafis, E.O. Campbell, P.T. Callaghan, Noninvasive imaging of water flow in plants by NMR microscopy, Protoplasma 173(3 4) (1993) 170 176. [26] C.W. Windt, Nuclear Magnetic Resonance Imaging of Sap Flow in Plants, University of Wageningen, Wageningen, 2007. [27] C.W. Windt, F.J. Vergeldt, P.A. DeJager, H.V. As, MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco, Plant Cell Environ. 29(9) (2006) 1715 1729. [28] V. Sarafis, H. Rumel, J. Pope, W. Kuhn, Non-invasive histochemistry of plant materials by magnetic resonance microscopy, Protoplasma 159(1) (1990) 70 73. [29] J. Pope, V. Sarafis, NMR microscopy, Chem. Aust. 57(7) (1990) 221 224.