Introduction to optical imaging

Optical microscopy is widely used in the study of biological systems thanks to a unique combination of benefits. The near infrared to ultraviolet light used for imaging allows us to observe spatial features ranging from organelles within the cell, to the extracellular matrix around cells, to the interactions of tissues in organs. In addition, because this light is non-ionizing, it is safe for imaging live cells and tissues, offering the ability to follow the dynamics of biological processes in real time. Optical imaging can be divided into two broad categories: imaging with labels or label-free imaging. My laboratory works on both classes of imaging to improve the capabilities of optical microscopy for studying biological problems. 

When imaging with labels, an external probe, typically a molecule or a nanoparticle, is introduced to the sample and light is collected from the probe. The most common probes are fluorescent, meaning that after optical excitation, the probe emits fluorescent light that is detected and used to form an image. Probes are designed to target particular ligands on biomolecules under study, permitting the monitoring of specific molecules and their interactions. Yet, these molecular probes are not without their limitations, including finite specificity, meaning that probes can bind to off-target molecules; toxicity to cells; and difficulties with diffusion into thick tissues. Despite these challenges, fluorescent probes continue to be powerful tools that advance biological sciences. My laboratory is working on new imaging methods that advance super-resolution imaging methods using the quantum properties of single quantum emitters, such as fluorescent molecules and quantum dots. 

The difficulties of labeling can be avoided by using intrinsic optical properties of biomolecules in cells and tissues. If no external label is applied, then the imaging method is called label-free. Label-free imaging methods exploit some form of light–matter interaction that converts light used for illumination into light used to form an image of the sample. A significant motivator for label-free imaging methods is to avoid the toxicity and disturbance of natural biological behaviors. Methods used for label-free imaging are wide-ranging, from simple optical absorption, phase shifting, optical scattering, vibrational spectroscopy, nonlinear spectroscopy, and photothermal detection. In addition, biological samples contain a rich set of valuable fluorescent molecules that has proven particularly powerful. These methods provide valuable information for metabolic activity, intracellular dynamics, detection of pathogens, and digital pathology, to name a few. 

But significant challenges in label-free imaging remain. A major problem is that label-free methods trade the molecular target specificity of external labels for ubiquitous signals that are difficult to relate to specific biomolecules of interest. The Bartels lab develops several label-free imaging methods that improve our ability to image biological dynamics under difficult conditions and to improve the molecular specificity of label-free methods.  

Optical imaging also faces limitations in spatial resolution, spectroscopic information, imaging speed and throughput, accessible imaging volume, and imaging in scattering environments like tissue. Just as a foggy night reduces the distance light can travel and distorts the image of the Wisconsin State Capitol above, tissue forces light to scatter. And the deeper into the tissue one tries to probe, the greater the scattering. 

Further, biological processes occurring in these tissues unfold not just over spatial but also temporal scales, both spanning orders of magnitude. Observing molecular processes requires a resolving power that exceeds that of conventional optical imaging. While tools such as electron microscopy offer a high-resolution window, these instruments provide a myopic field-of-view and in general are more restrictive than optical techniques in terms of sample preparation, the capacity to characterize the volume of the specimen (~100 nanometers), and the ability to track temporal dynamics. As biological dynamics are driven by random fluctuations that enable the interactions of biomolecules, imaging tools play a vital function in unraveling the complexities of biological systems and will continue to play a central role in understanding disease etiology and pathology. 

The Bartels lab works on novel imaging technology development to address these deficiencies in imaging methods. We work to solve longstanding barriers in imaging and apply those advanced technologies to studying biological systems that can only be studied in limited ways with current technology.