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Biomedical Optical Phase Microscopy and Nanoscopy

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Preface

Living biological specimens, such as cells, tissues, or microorganisms, are microscopic dynamic objects, continuously responding to their environments and performing multiple processes by adjusting their three-dimensional sizes, shapes, and other biophysical features. Microscopy and nanoscopy of living specimens can provide a powerful tool for basic biological and biophysical studies, and for medical diagnosis and monitoring of disease progression.

Many living biological specimens such as cells in-vitro are transparent objects, and imaging them with conventional bright-field light microscopy fails to provide adequate contrast in the microscope image. For this reason, exogenous contrast agents such as fluorescent labels are widely used in biomedical microscopy. However, these exogenous agents are often cytotoxic and may influence the specimen behavior, especially in the long run. Additionally, fluorescent agents tend to photobleach which might limit imaging time and make signal quantification difficult.

Phase microscopy proposes a unique solution to the contrast problem. Phase is proportional to optical path delays of the light passing through the sample, and thus it captures information on the specimen structure and dynamics without using exogenous labeling.

This book presents a cutting-edge review of a variety of phase microscopy and nanoscopy techniques with emphasis on biomedical and clinical applications. The authors of each chapter are internationally renowned scientists, either phase microscopy and nanoscopy technology experts, or researchers who are interested in the biological and medical applications of these technologies. Based on this heterogeneous nature of the contributing authors, this book will not only be useful for researchers in the areas of biomedical engineering, electro-optics engineering, and nanotechnology engineering, but can also help biologists and clinicians who are interested in understanding the underlying principles and in learning about new biological and medical applications of conventional and novel phase microscopy methods.

As editors, we have invested efforts to order the book as a whole and the structure of each chapter in such as a way that the book is self-contained, with introductory subjects, followed by specific biomedical applications. Therefore, we expect that this book will be useful for researchers at all levels including advanced undergraduate students, graduate students, postdoctoral fellows, and established researchers.

The book is divided into four parts based on the historical order of the technology inventions, starting with the older and widely-used technologies and finishing with the newer ones. The four part of the books are: Phase Contrast Microscopy and Differential Interference Contrast (DIC) Microscopy, Digital Holographic Phase Microscopy, Advanced Interferometric and Polarization Techniques, and Phase Nanoscopy. The first chapters in each of the four parts of the book contain an introduction to the relevant basic optical principles, followed by more advanced chapters that describe state-of-the-art advances and new applications.

Part 1 of this book deals with conventional and widely used phase microscopy methods: phase contrast and DIC microscopy. Chapter 1 introduces the basic principles of phase contrast microscopy, the first phase microscopy method, proposed by Zernike in the third decade of the nineteenth century, an invention that gained him the Nobel Prize in Physics in 1953. The typical artifacts of this technique and some common applications are also reviewed in this chapter. Chapter 2 presents another widely used phase microscopy technique: DIC microscopy, invented by Francis H. Smith in 1947. Chapter 3 describes a practical method that is based on phase contrast microscopy for conducting long-term time-lapse observations of living cells. Chapter 4 reviews phase imaging methods for plant cells and tissues.

Part 2 of this book introduces quantitative phase microscopy performed by digital holographic microscopy (DHM). Chapter 5 explains the principles of DHM used for measuring the quantitative phase maps and their interpretation for the calculation of biophysical parameters of biological cells. Chapter 6 presents new DHM setups and various applications. Chapter 7 focuses on the problem of 2π ambiguities in the quantitative phase profile and the possible solutions. Chapter 8 presents compact and portable phase microscopic designs and demonstrates the analysis of living sperm. Chapter 9 focuses on super-resolution methods using synthetic aperture for a simplified lensless DHM setup, which is used as examining tools for red blood cells and sperm cells. Chapter 10 presents the application of DHM for analyzing particles or live cells in microfluidic devices. Finally, Chapter 11 presents low-coherence DHM-based methods for tracing the internal motion in live cells and tissues, and demonstrates its applications for mapping functional motion in tumors and assessing drug effects.

Part 3 of this book focuses on advanced polarization and interferometric methods of phase microscopy, which are based on DHM introduced in Part 2 of this book, polarization microscopy, or a combination of both. Chapter 12 presents the tomographic phase microscopy technique in which the sample phase is captured using multi-viewpoint interferometric method, and then the data are processed into the three-dimensional map of the refractive index of the sample. Several demonstrations of cell imaging are presented. Chapter 13 discusses the phase-sensitive optical coherence tomography (OCT), an interferometric method for measuring the quantitative phase for a single-point or a line of points on the sample, and several possible applications. Chapter 14 presents various advanced interferometric and DIC methods that use polarized light to capture the phase of the sample. Finally, Chapter 15 presents the polarization microscopy, a label-free technique that exploits the inherent birefringence in living cells to visualize dynamics of cell organelles.

Last but not the least, Part 4 of this book introduces phase nanoscopy used to visualize nanoscale objects. Chapter 16 discusses the idea of breaking the spatial resolution limit in phase microscopy, including definitions of the theoretical limit of resolution. Chapter 17 presents a method for combining total internal reflection (TIR) and DHM for visualizing nanoscale objects. Finally, Chapter 18 presents a unique nanoscopy approach that is based on interferometric imaging of fluorescence molecules using self-interference.

We strongly believe that phase microscopy and nanoscopy in the fields of biomedical, electro-optical and nanotechnology engineering will continue to develop rapidly and become one of the most important and widely used tools in biology and medicine, while offering unique opportunities for new and exciting applications. We hope that this book will contribute to the development of the field by providing a balanced and self-contained presentation of the field from different perspectives.

Natan T. Shaked, Ph.D., Tel–Aviv University, Israel

Zeev Zalevsky, Ph.D., Bar–Ilan University, Israel

Lisa L. Satterwhite, Ph.D., Duke University, USA

Part 1

Phase Contrast Microscopy and Differential Interference Contrast Microscopy

Chapter 1 Phase Contrast Microscopy

Chapter 2 Differential Interference Microscopy

Chapter 3 Long-Term Recordings of Live Human Cells Using Phase Contrast Microscopy

Chapter 4 Phase Imaging in Plant Cells and Tissues

Chapter 1

Phase Contrast Microscopy

Sam A. JohnsonEditor: Lisa L. Satterwhite

Light Microscopy Core Facility, Duke University and Duke University Medical Center, Durham, NC

1.1 Introduction

Since the mid-1600s microscopes of increasing sophistication have opened a world invisible to the naked eye and are now ubiquitous and essential biomedical research tools. Magnification allowed observation of some objects and structures that were previously unknown and the capture of details impossible without the use of optics. This alone was transformative and opened entire fields of study.

But magnification is nothing without contrast—defined as the difference in intensity between two points divided by their average intensity—and many samples of considerable biological interest provide very limited contrast in homogeneous brightfield transmitted light microscopy. Phase contrast provides a means of extracting additional contrast from a variety of samples which provide little absorption-based contrast. Contrast can also be added by preparation and manipulation of the sample but this often involves fixing the sample, which can confer a considerable disadvantage in the study of biology and the dynamic processes it involves. Therefore, a method of observation of intact, live, and unlabeled samples is key.

Figure 1.1 shows the difference between a brightfield image and a phase contrast image of a typical thin transparent biological sample, a flat mammalian cell.

Figure 1.1 Comparison of a mammalian cell imaged with (A) brightfield and (B) phase contrast light microscopy.

As seen in Fig. 1.1, phase contrast [1–5] provides additional clarity and detail in live biological samples: more contrast is provided and finer structures can be visualized. Today, these abilities make it an indispensable tool for many who are interested in imaging diverse samples such as cultured cells, bacteria, thin tissue samples, and yeast. The significance and advance that this method of contrast formation provides merited a Nobel Prize in 1953 to Zernike for its development in the 1930s. Today, phase contrast is a very common feature of thousands of microscopes worldwide and can be considered an absolutely core technique of microscopy. This chapter will explain how phase contrast works and how it is done practically in standard microscopes; some typical uses of the technique are also presented.

1.2 How Phase Contrast Works

1.2.1 Basic Overview

Brightfield transmitted light microscopy has a fundamental weakness in the study of thin transparent biological specimens: they generally absorb little light to provide limited contrast by this means alone. The samples do however interact with light in ways other than absorption, and phase contrast is a method to exploit this and turn these interactions into observable contrast.

What does happen to light as it passes through transparent biological material such as the cell in Figure 1.1? Though transparent, the cell will cause diffraction and scatter of some of the light that passes through it. This process causes deviation and a phase shift of λ/4 between the small amounts of light that is deviated relative to the undeviated wave. A cell is far from homogeneous. The variety of components of the cell and the molecular, macromolecular, and organelle level structures into which they are arranged provide a complex landscape of changes in refractive index, and this variation will also have an effect on the light passing through the different parts of the cell.

Refractive index of a medium is defined as the ratio of speed of light in a vacuum to the speed of light in a medium.

(1.1)

A vacuum therefore has a refractive index of 1.0000, air 1.0003, water 1.33, cytosol 1.35, and the glass most commonly used to make optics 1.52. The refractive index of a medium is an aspect of the interaction between the light and the electrical and magnetic susceptibilities of the medium. Overall the cell has a different refractive index than the surrounding medium. Finer scale differences within the cell provide observable subcellular resolution: different constituents such as lipids, proteins, and nucleic acids and different concentrations of these in an organelle or region produce local changes in refractive index. In a transmitted light image, some of the light will have passed through a cell and will have interacted with this complex refractive index topology and have been affected. Both the occurrence of diffraction of the light and the refractive index differences produce phase shifts which can be exploited to produce contrast.

This and the consequent means of exploiting the effects in a phase contrast microscope are most easily explained pictorially with a series of annotated diagrams.

Figure 1.2 shows schematically a phase shift by a sample. Reducing the sample to a gray box that represents an object, such as a cell, of a different refractive index to its surroundings and having a propensity to diffract and deviate some of the light we see light that passes through it becomes phase shifted relative to a reference wave that passes around the object unaffected. The phase shift has two components: (1) diffraction causes a λ/4 phase shift and (2) the difference in refractive index and optical path length changes the speed of the wave and so the transit time across the object relative to the surround which causes a typically small phase shift between the deviated wave passing through the sample and reference surround wave. The object here has a higher refractive index, a lower speed of transmission of the wavefront, so the phase is retarded slightly. It is noted that no difference in amplitude is produced—there is no absorption—and so no contrast is provided by this means alone. Our eyes and digital cameras are insensitive to phase or differences in phase of light, and we are unable to distinguish the object from the surrounding background without further action. (Objects such as our limiting case example which produce a phase shift but no absorption are called phase objects; at the other extreme, objects which produce strong absorption but little phase interaction are termed amplitude objects but of course nearly everything really lies somewhere between these two extremes.)

Figure 1.2 Phase shift introduced by a sample.

The change in phase presents some possibility for interference between the light that is shifted and that which is not. An observable difference in intensity could be produced in this way. To provide a very simple example consider the wavefronts occurring in Figure 1.2. Of the light illuminating the sample and surrounding, most of the light is undeviated (the zeroth-order light) and has no phase shift. Some of the light passes through the sample and is diffracted and phase shifted, by an amount of around λ/4. The precise magnitude of the phase shift is the sum of the phase shift from diffraction (λ/4) and the effect of a difference in the optical path length—the product of refractive index and thickness.

(1.2)

(1.3)

The relationship between the optical path difference and the phase shift of a wave in radians is

(1.4)

Using examples of refractive index of the cytosol (1.35) and culture medium (1.33) and a range of thickness of the cell up to 10 μm, it can be calculated that the phase shift from optical path length differences are generally smaller than λ/4. Figure 1.3 shows the phase shift between the undeviated surround wave and the deviated wave shifted by around λ/4.

Figure 1.3 Phase shift between the diffracted wave and the surround wave.

Because of the limited extent of the phase shift (shown in Figure 1.3A) and the difference in amplitude of the two waves (which to underscore is due to only a small fraction of the light being diffracted rather than this light being attenuated) the difference between the observed interfered output (the particle wave) and unaltered surround wave is very minimal. The small phase difference between the S- and the P-waves is shown in Figure 1.3B and the amplitudes of the S- and the P-waves are essentially the same so we are still not provided with contrast to distinguish the object from background. This is essentially the state in brightfield illumination. How could a greater magnitude of interference be produced?

If we are able to produce an additional relative phase shift of λ/4 by advancing the S-wave, the total will be ~λ/2 (Figure 1.4C), thus presenting the maximal destructive interference potential. If we are also able to make the two waves of more equal amplitude, the efficiency of extinction through interference will be higher. This situation providing very high extinction is shown in Figure 1.4.

Figure 1.4 Phase shift between the diffracted wave and the phase shifted surround wave.

Should you prefer these concepts in vector diagram form where phase is presented by angle (retardation is clockwise, an advance is anticlockwise) and the amplitude by length of the line—the two situations of modest phase shift (Figure 1.3) and very high extinction (Figure 1.4) in phasor diagrams are shown in Figure 1.5.

Figure 1.5 Phasor diagrams of the phase shifts in Figures 1.3 and 1.4 .

How do we construct a physical optical arrangement to perform this contrast formation? The key point is the spatial separation of the phase shifted diffracted light and the reference waves. This is achieved by a structured pattern of illumination produced by a ring annulus in the condenser and a matching phase plate in the objective. Figure 1.6 shows a simplified schematic cross-section diagram of the arrangement in an inverted phase microscope. (The diagram simplifies the light path slightly to make it clear how this is achieved; the illumination in reality is not precisely a hollow cone but broader parallel wavefronts that are focused by the objective back to a ring pattern in the back focal plane.)

Figure 1.6 Schematic diagram of the arrangement of a phase contrast microscope.

A light source giving homogeneous illumination is used. This is typically a halogen bulb or LED providing Köhler illumination of the sample. The light that passes straight through without being diffracted (the zeroth order, undeviated, or reference waves) passes through the phase plate in a specific region (the conjugate region). The light that is diffracted changes angle and when collected by the objective passes through a different region of the phase plate (the complementary region). The phase plate is of a different thickness in these two regions and the difference in the optical path lengths (the product of refractive index (n) and thickness) produces a phase shift between surround and diffracted waves of λ/4. The undeviated light in Figure 1.6 passes through a thinner region of the phase plate and is advanced relative to the diffracted waves. This addition of a phase shift between the two waves adds to the difference in phase shift produced by the sample to enable intensity changes at the image plane through interference of the two waves.

The second function of the phase plate is to balance the intensity of the diffracted and reference waves, again achieved by means of them being spatially separated and passing through a different part of the phase plate. The unaltered reference wave passes through the ring on the phase plate, which is typically made partially opaque with a neutral density coating to attenuate this light to make it more comparable in amplitude to the diffracted light. Typically, around 75% of the surround light is absorbed at the phase plate to provide balance.

The two waves then interfere and a high contrast image is produced at the image plane that may be captured by CCD or CMOS camera, or viewed with extra magnification through eyepieces. Maximum extinction will be produced by regions or objects which produce a deviated wave that has a phase shift which when summed with the phase shift introduced by the phase plate produce a total shift of λ/2 and is of intensity comparable to the attenuated surround wave. A heterogeneous biological sample will also produce a range of phase shifts and extents of diffraction to have a range of contrast.

1.2.2 What Do You Actually See in a Phase Contrast Image?

The contrast added by exploiting phase effects within a sample clearly reveals many details not otherwise visible. With understanding of the method of phase contrast, it is easy to remember that darker regions of the image are not accounted for by absorption. The edge of a culture cell is clearly defined because of the difference between the propensity for diffraction and the phase shift in the cell and space surrounding it. But what are the biological structures or materials that give contrast within a sample? What of the organelles inside a cell—what is visible, what is not? With knowledge of the structure of a cell, it is clear that many familiar structures are visible—the nucleus, the nucleolus, and the vacuoles are clearly defined and identifiable. Other organelles are typically less clear but can be seen (e.g., mitochondria).

The simplest general explanation of the difference in contrast across a sample is the difference in optical path length (as defined earlier, this is the product of refractive index and thickness) at different points. Organelles and fine structures within the cell produce small local differences in refractive indices but because the thickness of the cell is also involved in the optical path length, the contrast distribution cannot be simply interpreted in terms of the refractive index. Often ruffles are visible in cells (see Figure 1.1), and these could be interpreted as differences in thickness of the cell with a relatively homogeneous refractive index of the cytosol but the precise contribution of the two components of optical path length are never known for sure.

The regions that appear darkest in a positive phase contrast image are those that present the extent of diffraction and phase shift from optical path length difference to the surround that produce a total phase shift offering highest extinction. Regions with a total phase shift differing from this maximum will appear lighter. It is not always trivial to attribute differences in intensity to particular directions of phase shift from optical path length differences since large, say, retardations may produce the same effect as a small advance. Indeed, thick objects with large optical path differences could produce a phase shift of more than one wavelength.

Even in the absence of linear direct quantitative attribution between levels of contrast observed, the magnitude of the phase shift that produces the contrast and the precise biological materials or subcellular structures that account for the contrast, the definition produced by phase contrast is extremely useful.

1.2.3 Positive and Negative Phase

The principles discussed earlier were explained in terms of positive phase contrast which is the most popular implementation in biological applications. Positive phase contrast involves retardation by the sample, and advance of the surround wave by the phase plate to produce λ/2 total relative phase shift for maximum extinction. A negative phase plate can be used and the principle of contrast formation is essentially the same except the phase plate produces a relative shift between diffracted and surround light in the opposite direction. In negative phase contrast, the surround wave is retarded relative to the diffracted wave and so regions of phase retardation within the sample produce positive interference and appear brighter than the background. It is somewhat a matter of sample-specific taste whether positive or negative phase gives better results. Generally, positive phase contrast is found to be more clear and these objectives are more commonly available. Figure 1.7 shows the difference in the phase plates, directions of phase shift of the S-waves, and examples of images of the same mammalian cell produced by positive and negative phase contrast.

Figure 1.7 (A) Positive and (B) negative phase contrast.

1.2.4 Halo Artifacts

Phase contrast images have a ring around the larger objects—for positive phase contrast this is a ring of bright light that surrounds the object, for negative phase contrast it is a dark ring that is between object and background. What causes the phase halo and why is it only seen around the outer edge of large objects? The halo is caused by light which is diffracted and phase shifted but not diffracted by a magnitude or direction that causes it to pass through the central part of the phase plate. The phase ring is normally made to be slightly larger than the conjugate size of the annulus so it efficiently accepts the surround light after the slight aberrations in the optics act to expand the size of the illumination ring. This makes the system slightly more susceptible to halo than would be expected. Large objects with lower spatial frequencies diffract the light at a lesser angle than finer scale structures represented by high spatial frequencies, so the light that comes from large objects accounts for the majority of this phase shifted light that passes incorrectly through the ring of the phase plate. Because this light is phase shifted by the phase plate in the wrong direction relative to the surround wave, no means of destructive interference are produced for this light and so a high intensity region is produced. The halo has both good and bad properties: good because it provides a strong intensity change around the edge of the cell, highlighting this region; bad because the spatial accuracy of the definition of the edge is reduced by the overwhelming contrast and because the relationship between intensity and optical path length differences is lost in this part of the image.

Shade-off is a related artifact in large homogeneous objects. The center of such objects appears brighter in positive phase contrast because this light is little deviated and so, just as in the halo, not acted upon by the phase contrast system to produce attenuation.

1.3 How to Acquire Phase Contrast Images

1.3.1 What Components Are in a Phase Contrast System?

Good news: only two extra parts are required over brightfield and the general layout remains compatible with other standard transmitted and epi-illumination imaging techniques and modalities. As described earlier, phase contrast is based on a ring-shaped illumination and a matching phase plate, most commonly in the objective itself. The illumination can be achieved by introducing a mask in the aperture plane in the condenser. These are simple metal disks with different sized rings; larger rings are used for higher power lenses. Most commonly manufacturers refer to phase condenser elements with Arabic numbers such as phase 1, 2, and 3 (Roman numerals are generally used for DIC).

The objective needs to be one specifically designed for phase contrast, but this typically now adds little cost to the optic. If an objective without a phase plate is used even with the phase condenser element, an image like brightfield will be obtained and represent the equivalent of Figure 1.3. Objectives capable of phase contrast generally have green writing and indicate which condenser phase ring position offers compatibility. The physical diameter of the ring in the phase plate decreases with increasing magnification and Numerical Aperture (NA) of the lens and generally covers about 10% of the area of the objective.

1.3.2 General Alignment

The components are easy to align. No rotary alignment between components or the samples is necessary in setting up the scope and only a few adjustments are necessary to get a phase image of optimal quality making the technique quick and simple to use.

Nearly all modern microscopes have a transmitted light path employing an adjustable condenser giving Köhler illumination and this is the starting point for phase contrast. It is important to establish Köhler illumination under brightfield for the objective to be used to ensure the condenser is centered and positioned so as the image and illumination planes are physically optimally arranged and homogeneous illumination be provided.

The correct phase ring within the condenser needs to be selected for the chosen phase objective. Commonly, when a phase ring position on a condenser is selected, the condenser aperture is moved out of the path or fully opened. If this is not the case and the condenser aperture is not opened to the diameter of the phase ring, all illumination will be blocked.

A color filter (most commonly green) may be included in the path to ensure the illumination reaching the sample is monochromatic or of a small spectral range. This helps to produce maximum contrast by ensuring optimal phase shift by the phase plate; if white light is used a range of phase shifts around λ/4 will be produced as described in Eq. (1.4). Defined color illumination also reduces the degradation of the contrast and resolution in the image from chromatic aberrations and allows inexpensive achromat lenses to be used.

1.3.3 Phase Ring Alignment

The most critical alignment in phase contrast is the coincidence of the illumination ring and phase plate in conjugate planes at the condenser aperture and back focal plane of the objective, respectively. Figure 1.8 shows the impact of misalignment of the phase ring and the consequent degradation of image contrast and quality. The image formed with misaligned phase optics is less clear because the misaligned illumination is not adequately attenuated by the phase plate, so it is bright and the imperfect contrast formation overwhelmed.

Figure 1.8 Phase ring alignment and an example of an image produced with a misaligned phase ring.

The phase ring can be aligned by viewing the back focal plane of the objective where the phase ring is present, and the annulus giving ring illumination is conjugate. This can be most easily viewed by removing one of the eyepieces. Magnification can be provided by using a Bertrand telescope but it is not impossible to align without this tool, especially for low power lenses. Removing the sample may make this alignment easier so the diffracted rays are not present to lessen the definition of the illumination ring. The sample was present when the images of the back focal plane of the objective were captured for Figure 1.8. The diffracted rays can be seen across the complementary region, but the illumination ring is still visible in the presence of these waves for this sample.

The condenser will have at least two screws giving fine adjustment of the position of the ring to allow superimposition. It is important to remember that adjustment screws in the condenser are not the same as those providing centration adjustments for Köhler alignment. Often once a ring is aligned for an objective, it tends to be fairly stable and may not need adjusting with every use but the alignment is very quick to test to make sure it is correct. The phase plate in the objective is virtually always fixed and nonadjustable.

1.3.4 Adjustability

Phase contrast requires fewer setup steps than many other contrast methods for transmitted light imaging. In most cases for a particular objective, once it is properly set up, there essentially are not any adjustments to make. Different samples present a substantial range of extent of phase shifts and diffraction. To cope with this range, different objectives can be used to find an optimal balance of the amount of surround light that needs to be blocked by the phase plate (based on the extent of diffraction by the sample). But these are generally different complete objectives so the cost of acquiring this variability is fairly significant.

1.4 Experimental Uses

Phase contrast is one of the most popular, versatile, and widely used transmitted light contrast techniques used today. It is difficult to represent adequately the breadth of uses in the biomedical research community, but this will be attempted by presenting some general classes of examples.

1.4.1 Cell Culture Models

Cell culture models are a very common research tool, and phase contrast is ideally suited to the routine observation of these cells. Simple and relatively inexpensive inverted microscopes with low magnification or higher magnification long working distance dry lenses with phase contrast optics are situated in nearly all tissue culture facilities. They provide a quick and effective means of assessing the health and density of the culture cells in the plastic culture dishes and flasks used. The key advantage of phase microscopy for this purpose (as well as simplicity and low cost) is the ability to image through relatively thick optically imperfect plastic. The plastic used in culture vessels tends to present relatively little absorption and local differential phase shift of the light but significant interaction (principally birefringence) with polarized light meaning techniques such as DIC are not achieved with good results without specialized versions of these optical contrast methods. See Chapter 3 for excellent description of how phase can be used in live cell time-lapse studies.

1.4.2 Image Analysis

Threshold-based image analysis routines allow quantification of a broad range of features of samples. Examples include automated counting of cells, measurement of areas, and tracking the movement of live cells in a time lapse. Thresholding involves physically identifying an object by a difference in intensity between the object of interest and background. Phase contrast has two advantages for certain uses of this. The first advantage is that it provides large contrast—the difference in intensity between the cell edge and the background—making identification easier and more robust than with the use of brightfield images. Anybody who has ever attempted any image analysis with biological samples using threshold-based identification will know that with more contrast available, the easier and more accurately one can pick apart the objects of interest and the task can range enormously in difficulty with, among other factors, the level of contrast available. Typically, transmitted light microscopy does not provide as high a level of contrast as does fluorescence because the background is bright, whereas it is dark in fluorescence and so the ratio of intensities is much larger. But the enhanced edge contrast provided by phase is adequate for identification of the edge of cells thus providing a very useful tool. The second advantage of phase contrast is the enhanced edge definition is provided symmetrically around the cell. With DIC (see Chapter 2), enhanced edge contrast is also provided but the shadow effect means it is bright on one side of the cell and dark on the other. Our visual system has no problem understanding this arrangement but an image analysis process looking only at differences in intensity needs to be smarter than one analyzing a symmetrical pattern. Figure 1.9 shows the ability of easy identification of a cell by phase contrast making it ideal for uses such as counting and tracking cells. The automated outlining (third row) of thresholded regions (second row) of a certain size range is produced. The lower contrast in brightfield does not allow easy contiguous identification of the cell perimeter. With DIC, the shadow gradient presents a significant issue.

Figure 1.9 Threshold-based cell identification in (A) brightfield, (B) phase contrast, and (C) DIC.

1.4.3 Fluorescence Overlay

As with other transmitted light microscopy techniques, phase contrast images can easily be overlaid with fluorescence images providing a general morphology of the sample on which the specific fluorescence labels can be interpreted. The transmitted and fluorescence images are taken sequentially using the same camera and digitally overlaid. As with many transmitted light and fluorescence overlays, the best results are often produced by scaling the transmitted image so that it is relatively dark so as not to overwhelm the fluorescence that is to be overlaid. Phase also provides a high contrast method to focus on and select live samples avoiding the use of phototoxic intense fluorescence illumination when no information is obtained with it.

Phase optics has a minor effect on the fluorescence image. Obviously, the ring annulus in the condenser is irrelevant for epi-fluorescence configuration and hence presents no issue. The phase plate is normally fixed in the objective and so fluorescence emission light passes through it. The light that passes through the phase ring itself will be attenuated, but the overall effect is quite minor but can be significant in very demanding applications where the loss of a few percent of the signal is noticeable. The light will also be partially phase shifted by the plate; since this is not coupled with spatially arranged illumination this is of limited consequence.

Confocal microscopes are rarely equipped with phase optics. Point scanning confocals could be affected a little more seriously than widefield as the beam pivots near the phase plate and a coherent light source is used so the aberrations could be more apparent. Also, the means of phase contrast formation described here is not compatible with a point-based assembly so a phase contrast transmitted correlate to a confocal image cannot be built with a nondescanned transmitted photo multiplier tube (PMT). DIC is typically used for transmitted light contrast overlay on a confocal, and this works well in point scanning with laser illumination that provides polarized light. DIC also offers the advantage of allowing the removal of the analyzer and Wollaston prism offering maximum photon collection in fluorescence imaging