The advent of fluorescence microscopy has been a major step forward in the study of living cells. Leveraging the characteristic emissions of excited biological fluorophores, such as fluorescent proteins, it is possible to gain insight on cell structure and function (Figure 2). Following traditional fluorescence microscopy has been the development of multi-photon methods, where fluorophores are excited by two or more photons 35. Multi-photon absorption is achieved with a single pulsed laser focused to a diffraction-limited spot on the specimen. With higher peak power, there is an increase in probability for multi-photon absorption leading to fluorophore excitation. Two-photon fluorescence is depicted in Figure 3a. To meet the excitation energy in this case, two 800 nm photons are used. One 400 nm photon is of equivalent energy, as can be used in single photon excitation, but with multi-photon methods only the area of the laser focus on the specimen is excited. Due to more focused excitation, there is a lower overall phototoxic effect. Also, as scattering of longer wavelength photons is less, multi-photon methods have deeper penetration when compared to single photon excitation.

In STED (Stimulated Emission Depletion) microscopy 3637, two pulsed lasers are used in tandem to break the diffraction barrier. The first laser pulse has a wavelength that excites fluorophores, and is immediately followed by a second laser pulse that depletes fluorescence. The fluorescence depletion is achieved as the wavelength of the second laser is tuned to be longer than the fluorescence emission. Absorption of a photon from the second laser induces electrons to drop to a lower energy level (stimulated emission) preventing typical fluorescence. The difference in area of the two focused beams leaves only a very small area from where fluorescence is detected. This area is smaller than a diffraction-limited spot. Using STED, images have been captured with a resolution of ~30 nm 35. In another study using rhomadine amide and a photoswitching technique, a resolution of 15 nm was achieved 38. In 3940, an optical trapping system was used to make angstrom resolution measurements of pair based stepping of RNA polymerase, and thus has established an important resolution benchmark in molecular biology.

Electron microscopy has offered a resolution of ~5 nm for imaging biological tissue 41. However, to prepare a sample to be imaged by an electron microscope is a rigorous process that does not allow for imaging of live samples 42. One common sample preparation technique is cryofixation, which is a high pressure and deep freezing technique that results in contrast in electron microscopy 43. Even though the samples are no longer viable, electron microscopy has provided invaluable insights on the structural details of organelles and membranes 44.

Atomic force microscopes do not acquire information optically, but rather by recording intermolecular forces between a probe tip and a surface. The primary information acquisition component of an atomic force microscope is a cantilever with a nanometer-scale silicon tip. The tip is brought in close proximity with the sample and the deflection of the cantilever due to Van der Waals forces is recorded to generate a contour map of the sample surface 4546. In comparison with an electron microscope, the sample does not need any special treatment that would actually destroy the sample and prevent its reuse. However, using contact or tapping mode of an atomic force microscope, which impinges on the sample surface to acquire measurements such as strain, can mechanically damage cells and tissue. Other probe microscopy techniques include scanning tunneling microscopy and near-field scanning optical microscopy 47.

Fluorescence microscopy is often used in systems biology and there is a strong push for the development of high throughput methods. In the application of genome-wide RNAi screens to document the phenotype for each suppressed gene 4849, there can be millions of images from a single screen which can amount to several terabytes of data 50. It is systems biology modeling that relieves the bottleneck of processing this large amount of RNAi screen image data by providing an efficient means of classification. With high throughput microscopy there is much more data generated than can be annotated or evaluated manually, and so developing a fine-tuned and efficient classification model is paramount for unlocking the potential of high throughput methods.

As seen in Figure 2, fluorescent proteins can be used to visualize many functional and structural aspects of cells. Using multi-photon methods as well can provide insight on cell structural and biochemical changes 51. Multi-photon methods have a wide array of applications including in vivo brain imaging in animals (Figure 3) 525354, where cortical micro-architecture has been investigated with single cell resolution 55. Electron microscopes have been used to elucidate macromolecule structure 41. As for cancer applications, atomic force microscopes have been used to monitor the super-coiled state of DNA, which is preferential to the binding of the tumor suppressing protein p53 56. Also in regard to detecting levels of p53 in cells, fluorescence microscopy has been used to determine the effectiveness of oncolytic adenoviruses. Specially designed oncolytic adenoviruses target cancerous tissues and are programmed to replicate if the cellular p53 level is low. Viral oncolytic therapy is an intense research area for cancer treatment and as microscopy techniques advance so will the ability to assess the effectiveness of viral vectors for tumor ablation 5758.

It has been long held that the wave nature of light imposes a seemingly fundamental limit on the resolving power of a microscope. The limitation was approximately half the wavelength of visible light or 200 nm. Recently, there has been over a 10-fold resolution improvement with advances in microscopy 3538. However, optical techniques have limited penetration as light readily scatters in tissue. This can be partially ameliorated by using more powerful lasers, but this in turn can lead to increased photobleaching effects which can limit the amount of time that an experiment can run.

Microscopy, as with other modern imaging techniques, has become ever more dependent on software for image acquisition and analysis. Imaging technology can be enhanced or limited by the software it is coupled with. Table 2 contains an overview of current microscopy image analysis software. With advances in acquisition algorithms and optics holographic microscopy has been achieved, by which full three-dimensional information can be acquired in a single image 5960. As a result, volumetric time series data can be collected without the need of changing focus and scanning multiple z-planes.

Ultrasound imaging entails moving a hand held probe over the patient and using a water-based gel to ensure good acoustic coupling. The probe contains one or more acoustic transducers and sends pulses of sound into the patient. Whenever a sound wave encounters a material with different acoustical impedance, part of the sound wave is reflected which the probe detects as an echo. The time it takes for the echo to travel back to the probe is measured and used to calculate the depth of the tissue interface causing the echo. The greater the difference between acoustic impedances, the larger the echo is. A computer is then used to interpret these echo waveforms to construct an image 61.

Ultrasound has had a tremendous impact in cardiology. As seen in Figure 4, the use of ultrasound can enable the coupling of anatomical and strain information of the heart 62. Going from the organ level to the molecular level has been made possible by advances in microbubble manufacturing. Microbubbles themselves are several micrometers in diameter and are intravascular tracers 63. Ligands can be attached to microbubbles to make them target specific 64. Many clinical applications of contrast enhanced ultrasound, such as monitoring angiogenesis and inflammatory response, rely on ultrasound detection of microbubbles that contain gas. Since microbubbles are confined to the vascular space, they are useful for targeting antigens expressed on endothelial and blood cell surfaces 63. Smaller nanoparticle based contrast agents are also available that are capable of extravascular migration in regions of vascular injury or regions where vascular permeability is abnormally high. Ultrasound can also cause a mechanical interaction with microbubbles, leading to their destruction and the subsequent release of therapeutic compounds 65.

In tumor and angiogenesis models, surface expression of α_vβ₃ has been demonstrated to be a strong ligand for targeting endothelial cells in angiogenic vessels 63. In order to image this surface expression, microbubbles were conjugated with peptides that bind to α_vβ₃. These microbubbles have been shown to have a binding preference to the endothelial surface of Fibroblast Growth Factor (FGF) stimulated neovessels. The extent of neovascularization in a matrigel model matched the image enhancement in ultrasound images to a large extent. Thus, ultrasound imaging served to help validate this experimental model for angiogenesis 63.

The signal-to-noise ratio for ultrasound images is much lower with nanoparticles than those using microbubbles. Although microbubbles are restricted to the vascular space, this can be an advantage since it minimizes potential signal interference from nonvascular cells 63. As with other molecular imaging techniques, there is an inverse relationship between sensitivity and resolution for contrast enhanced ultrasound. The relative rate of unbound tracer clearance is also an important issue that determines temporal resolution. In this regard, with clearance time within minutes, microbubble tracers are ideal 63.

Intrinsic differences in X-ray absorption among water, bone, fat, and air provide contrast in Computed Tomography (CT). In CT, a low energy X-ray source and a detector rotate around the subject, acquiring volumetric data. The detectors are typically Charged Coupled Devices (CCD) and act to phototransduce incoming X-rays 66. For animal studies, microCT machines can be used which typically operate with higher energy X-rays when compared to human scanners. The increase in energy improves resolution, but exposes the specimen to more ionizing radiation which has adverse health effects.

CT has relatively low soft tissue contrast for tumors and surrounding tissue, but with iodinated contrast agents organs and tumors can be detected 29. As a result, incorporating iodine into new probes for CT imaging may be necessary. Furthermore, to detect a tumor or other target there must be sufficient site-specific accumulation of probes to result in attenuation of X-rays. With differential attenuation of X-rays, the target can be more readily delineated 7.

CT can be used to image lung tumors and bone metastasis, given its fast imaging time and high spatial resolution. High throughput techniques using microCT have been used for phenotyping large numbers of transgenic mice and detecting macroscopic abnormalities 64. In 67, the co-registration of microCT images containing tumor structural details with bioluminescence images allowed for the study of cell trafficking, tumor growth, and response to therapy in vivo. This image analysis method could potentially be used for assessing hematological reconstitution following bone marrow transplantation.

As seen in Figure 5, microCT imaging and volumetric decomposition were used to provide insight on trabecular bone microarchitecture 68. The bone samples were decomposed into individual plates and rods, and this imaging and processing scheme has been successfully applied to anatomic sites such as the proximal femur, proximal tibia, and spine. Several key morphological features of trabecular bone architecture were studied: plate and rod size, thickness, number density, and orientation. With this level of detail, it was determined that trabecular plates play an essential role in determining the elastic properties of trabecular bone 68. Assessing such properties can be important for gauging bone health in conditions such as osteoporosis, and for designing viable replacement tissue in tissue engineering applications 69.

A key advantage of CT is its high spatial resolution, 12 – 50 μm 2970, which is needed to visualize fine anatomical details. CT can also be combined with functional imaging technologies that provide dynamic and metabolic information. The radiation dose of CT, however, is not negligible and this limits repeated imaging in human studies due to health risks 64.

Magnetic Resonance Imaging (MRI) is achieved by placing a subject in a strong magnetic field, typically 1.5 or 3 Tesla for human scanners, which aligns the hydrogen nuclei spins in a direction parallel to the field. A Radio Frequency (RF) pulse is applied to the sample which causes the spins to acquire enough energy to tilt and precess, where an RF receiver can record the resulting signal 71. After the removal of the RF pulse, the spins realign parallel to the main magnetic field with a time constant of T1 which is tissue dependent. Signal strength decreases in time with a loss of phase coherence of the spins. This decrease occurs at a time constant T2 which is always less than T1. Magnetic gradients are used to localize spins in space, enabling an image to be formed. The difference in spin density among different tissues in a heterogeneous specimen enables the excellent tissue contrast of MRI 71. MicroMRI follows the same principles, but a much higher magnetic field strength is used for animal studies. Increasing magnetic field strength improves resolution, but can disturb the visual system and lead to peripheral nerve stimulation.

Functional Magnetic Resonance Imaging (fMRI) is a modality used to image brain activity in response to specified stimuli. When a stimulus solicits a response from a certain area of the brain, metabolism in that region increases. Metabolic demand leads to an increase in blood flow and more oxygenated hemoglobin in the region. As the supply of oxygenated hemoglobin exceeds the metabolic demand, the concentration of oxygenated hemoglobin increases. The balance between oxygenated and deoxygenated hemoglobin is altered leading to a change in image contrast. To detect a change, the image is compared with baseline measurements. Typical cortical activation leads to a 1 – 5% increase in image intensity 72.

Magnetic Resonance Spectroscopy (MRS) is an emerging imaging and biochemical analysis technique in biomedical science. It combines the analytical ability of Nuclear Magnetic Resonance (NMR) to identify biochemical species with the capabilities of MRI to isolate individual voxels which are three-dimensional pixels. MRS employs chemical shift imaging to localize spectra for individual voxels 73. This is achieved by phase modulated RF pulses which eliminate signal contamination into neighboring voxels. When MRS is combined with MRI, concurrent anatomical and biochemical information is obtained (Figure 6).

The range of microMRI applications spans from purely experimental to preclinical. MicroMRI technology has been used to track stem cells, monitor immune cell proliferation, and describe embryological development 74. It has also been used to obtain three-dimensional high resolution representations of bone structure 75. MicroMRI has advanced to the point at which individual cells, and their organelles, can be imaged with spatial resolution of <4 microns. Images of a paramecium and a spirogyra alga were acquired utilizing a magnetic field of 9 Tesla, phase encoding in all three axes (which improves signal to noise), and Carr-Purcell echo refocusing (incorporation of multiple 90 degree spin echo pulses into the sequence to minimize signal loss due to sample inhomogeneity) 76.

Contrast agents have been developed with greater affinity for cellular and molecular targets. These include iron oxide particles (which have been used to label individual T cells), manganese ions (which act as a paramagnetic surrogate of calcium), and caged compounds. The latter involves chelated gadolinium surrounded by an enzyme substrate, which physically obstructs water molecules from approaching the gadolinium. When an enzyme cleaves the substrate, water is able to approach the gadolinium. This in turn reduces T1 and increases contrast. The caged-compound technique has been used to demonstrate regionalized in vivo gene expression in frog embryos whereas manganese ions have been used to trace neuronal pathways 74.

fMRI is used to study the functions of the living brain in a non-invasive manner. It has been shown with fMRI that different cognitive functions, such as attention, perception, imagery, language, and memory, elicit specific cognitive activation patterns in different regions of the brain. One common clinical use of fMRI is in the treatment of patients with brain tumors, and a primary treatment goal is to preserve functional brain tissue. fMRI is used to determine the functionality of brain tissue surrounding the tumor so that potentially harmful therapy can be directed away from critical areas 77.

Due to the ability of MRS to identify the presence of molecules within voxels, many studies have been devoted to using it to help diagnose cancer and characterize neoplastic tissue. Currently, MRS has been successfully employed in regard to brain, breast, and prostate cancer through identification of various biochemical markers of neoplasm in the imaged volume 7879. ¹H has been the element of choice because of its large abundance, but studies involving ³¹P and ¹³C appear promising. The latter has been used as an effective dynamic marker of metabolic processes through a hyperpolarization technique 80.

The two chief advantages of MRI are its excellent tissue contrast and lack of ionizing radiation 74. Improved signal-to-noise ratio and resolution can be obtained via a small receiver coil radius and high magnetic field strength. However, high magnetic field strength is problematic in human applications because of arising physiological effects such as nausea and visual abnormalities. Also, higher field strength leads to other technical challenges including an increase in the operating frequency, which potentially generates artifacts.

The main advantage of fMRI is its ability to non-invasively image brain. Since image contrast is achieved through the levels of oxygenated and deoxygenated hemoglobin, no external contrast agent is needed. However, due to the faster temporal resolution needed to acquire images of dynamic brain activity, spatial resolution is reduced.

Due to the ability of MRS to reveal the presence of particular biomedical molecules and compounds within an in vivo sample, it seems ideally poised for use in systems biology research. However, certain challenges must be overcome such as large voxel size, long sampling times, and questionable quantitative accuracy of assessing molecular concentrations 81.

In Positron Emission Tomography (PET), radioactive tracers are incorporated into metabolically active molecules and then injected intravenously. There is a waiting period while the metabolically active molecules are concentrated in the target tissue. The molecule most commonly used in PET is fluorodeoxyglucose (FDG), which has radioactive fluorine and is readily taken up by tumors. The radioactive tracer decays and produces two 511 keV gamma-rays, which result from the annihilation of a positron and an electron. The two resultant gamma-rays are emitted nearly 180 degrees apart and observed by detector rings. Figure 7 contains several sample PET images. The sensitivity of PET at detecting molecular species is relatively high, in the range of 10^-11 – 10^-12 M. For animal studies, microPET has a volumetric resolution of 8 mm³, while next generation scanners have over an 8-fold increase in resolution and a field of view that encompasses the whole body of a mouse 2982.

There are many radioactive tracers for PET that are used in different preclinical and clinical applications 7. The tracers that target specific tumors are essential for systems biology studies due to the information provided regarding metabolic activity 8384. Examples of small targeting ligands include ¹¹C-labelled N-methylspiperone and ¹⁸F-labelled spiperone for targeting dopamine receptors on pituitary adenomas 83.

PET is useful in systems biology studies related to bone metabolism 85 and metastasis. Bone metastasis is common for several cancers, including prostate, breast, and lung 86. ¹⁵O-labelled water can be extracted from the blood and used to assess tumor blood perfusion. Tumors are in constant need of nutrients from the blood, and tumor neovascularization provides a crucial lifeline for rapidly dividing tumor cells. The uptake of tracer into tissues is proportional to delivery, and so is a measure of perfusion 87.

PET can be used for measuring therapeutic effects on disease processes. Specific metabolic enzymes that are selectively expressed in prostate cancer cells constitute such a target. In 11, genes that were differentially expressed between early stage and late stage prostate cancer were studied. L-lactate dehydrogenase-A catalyzes the formation of pyruvate from S-lactase and was expressed at a high level in the late stage cancer cells. PET tracers based on this process would serve to validate this finding and may allow for the identification of prostate cancer metastasis 11.

PET is a highly sensitive, minimally-invasive technology that is ideally suited for pre-clinical and clinical imaging of cancer biology. By using radioactive tracers, three-dimensional images can be reconstructed to show the concentration and locations of metabolic molecules of interest 2. Since the study of cancer cells in their normal environment within intact living subjects is essential, PET is ideally suited for monitoring molecular events early in the course of a disease, as well as during pharmacological or radiation therapy. Furthermore, it can be used to acquire prognostic information and to image for disease recurrence 282.

PET spatial resolution is comparatively poor, and is limited by pixel sampling rate, the source size, and blurring in the phosphor screens of the detector rings. Another limitation of PET is that radioisotopes with very short half lives must be immediately injected after production. Due to the same decay type of the different radioactive tracers, it is only possible to trace one molecular species in a given imaging experiment or clinical scan 64.

Quantum dots (QD) are a class of polymer-encapsulated and bioconjugated probes that can fluoresce at multiple wavelengths spanning the visible spectrum. Larger quantum dots emit red light while smaller ones emit blue light. Quantum dots themselves are comprised of a semiconductor core, encased in another semiconductor material that has a larger spectral band gap. This construction enables fluorescence upon excitation. Quantum dots can be packaged in amphiphilic polymers and conjugated with targeting ligands for imaging applications 88. Under harsh conditions such as wide pH range (1–14), varied salt conditions (0.01 to 1 M), and a strong corrosive environment (1.0 M hydrochloric acid), quantum dots demonstrate extraordinary resiliency and sustained functionality 14.

Figure 8 shows the proliferation of human Mesenchymal Stem Cells (hMSC) that are labeled with quantum dots. After 22 days the quantum dots remained incorporated in the hMSCs. This study suggests that bioconjugated quantum dots are a viable probe for long-term labeling of stem cells 89.

Ligands on quantum dots can be tailored to target specific cancer lines. Quantum dots fashioned to target prostate cancer, QD-PSMA (Prostate Specific Membrane Antigen), showed active emission in the presence of C4-2 prostate cancer cells while other quantum dots did not 14. Quantum dots can also be used to passively target tumors since leaky tumor vasculatures retain more quantum dots than surrounding healthy tissue. Thus, by both active binding and passive diffusion, more quantum dots will be present near cancerous tissue 14. With the targeting capabilities of quantum dots there is potential for use as a delivery vehicle for therapeutic compounds. Delivery schemes can be based on the release of a therapeutic compound triggered by ligand binding 13141690. As an example of a drug delivery application, in 91 quantum dots with cadmium sulfide were used as chemically removable caps inside mesoporous silica nanospheres to prevent the premature release of drug molecules. Targeted release of drug molecules was mediated by disulfide bond-reducing agents. Quantum dots could also be used in photodynamic therapy by which there is an energy transfer from the quantum dots to target cells, leading to the generation of reactive oxygen species, and thus potentially inducing apoptosis 9293. One limitation of such a therapy in vivo would be reliable and localized energy transfer to ensure the destruction of specific cells.

As applied to bacteriophage development, quantum dots can be multi-purpose by validating the design model as well as showing the effectiveness of tumor targeting 34. One design model for bacteriophages with quantum dots is based upon the characteristics of quantum dots themselves. These are namely durability due to the co-polymer shell and flexibility due to the possibility of several different ligands. Experiments have been conducted with quantum dot embedded bacteriophages in both in vitro and in vivo with the goal of destroying cancerous tissues. Iteratively designing and creating bacteriophages is an example where quantum dots provide both the effective targeting means, but also the validation of the design model due to visualization of ligand binding 34.

Information acquired by using quantum dots are constrained by the physical limits of fluorescence microscopy, since that is the imaging technique typically used when detecting emissions from quantum dots. There have been some studies using quantum dots in electron microscopy, which has an order of magnitude higher resolution than light microscopy 90. The quantum dots themselves experience "blinking," as in each quantum dot randomly switches from on to off. Fortunately, the fluorescence of a bound quantum dot is stronger than of an unbound quantum dot. Still, the randomness of "blinking" imposes some limitations on applications requiring single molecule detection as well as on applications requiring quantification of total fluorescence 13. Using two different color quantum dots, single molecule imaging has been achieved by co-localization on target molecules 94.

Nanoshells are a class of metal nanostructures consisting of a dielectric silica core surrounded by a very thin metallic shell. By varying the core to shell ratio and the overall size of the nanoshells, strong scattering properties can be achieved that result in resonance wavelengths generating heat 15. See Figure 9 for cross sectional views of a nanoshell. Fabricating nanoshells with specific antibodies provide a means for scattering based molecular imaging 1595, which provides molecule specific contrast on the nanometer scale.

As seen in Figure 10, nanoshells can facilitate tumor ablation. In another cancer related study 15, nanoshells were used as contrast enhancers to image HER2 expression, a clinically relevant marker in human breast adenocarcinoma cells. Gold nanoshells were fabricated and tuned for Near Infrared (NIR) imaging. Then, the nanoshells were exposed to HER2 (specific) or IgG PEG-ylated (non-specific) antibodies to facilitate targeting of cultured human mammary adenocarcinoma cells. A microscope equipped with a bright field and dark field were used to evaluate cell viability. In vitro photothermal nanoshell therapy was performed and silver staining demonstrated the tissue targeting specificity of HER2 nanoshells as well as the non-specificity of IgG nanoshells. In addition to mediating photothermal destruction of breast cancer cells in vitro, it was demonstrated that NIR absorbing nanoshell bioconjugates can provide molecular specific optical contrast enhancement without cytotoxicity 15.

Optical imaging with nanoshells offers the potential for non-invasive, high resolution in vivo imaging at relatively low cost 15. Scattering based optical imaging technologies rely on inherent changes in indices of refraction. Strategies that depend only on the intrinsic optical contrast within tissue have proved clinically valuable in some screening applications. However, such techniques are not sensitive enough to resolve an image based on disease biomarkers 15. In cancer, when early detection is critical to reducing morbidity and mortality, the use of molecule specific contrast agents provides the ability to optically sense and image abnormalities long before pathologic changes occur at the anatomic level 15. In the future, nanoshells may provide excellent contrast for other imaging modalities such as CT 96.