What is light microscopy?
The primary function of a microscope is to enable us to observe objects that are too small to be seen with the naked eye. These objects are typically extremely tiny, making it necessary to magnify them to be visible. Much like a magnifying glass, magnification is one of the fundamental aspects of a microscope. While a magnifying glass may only be capable of magnifying objects up to a certain level, such as 5X or 10X maximum, a microscope can easily achieve 100X, 2000X, or even higher magnifications.
However, magnification alone is not sufficient. When we magnify an object hundreds or thousands of times, there is a risk of the image becoming blurry. To illustrate this point, consider taking a picture with your mobile phone and then zooming in. As you zoom in, the picture gradually becomes blurry. This phenomenon also applies to microscopes and is closely associated with the concept of resolution. Resolution, together with magnification, is one of the most important properties of a microscope. When a microscope enables us to visualize extremely small objects, it must maintain a high level of resolution. Resolution refers to the microscope’s ability to distinguish and separate between two closely positioned points.
In addition to magnification and resolution, intensity and/or color differences create image contrast when imaging specimens using a light microscope. This contrast is crucial because it allows us to discern individual features and details of the sample. Contrast is defined as the disparity in light intensity between the image and the surrounding background. Generally, the human eye requires a minimum contrast value of 0.02 (2 percent) to perceive differences between the image and its background.
The magnification and resolution of a modern light microscope primarily depend on the objective lens, which is a fixed property. However, the overall configuration of the microscope can impact or potentially diminish these properties.
On the other hand, enhancing the contrast in microscopy can be achieved through various technologies, each with advantages and disadvantages.
In our laboratory, we utilize TIRF (Total Internal Reflection Fluorescence), IRM (Interference Reflection Microscopy), and iSCAT (Interferometric Scattering) techniques to enhance the contrast of the microscope. These methods allow us to improve the visibility and differentiation of features in our study specimens.
We employ point-spread-function fitting to track motors stepping along microtubules with high spatial precision in single-molecule experiments.
TIRF Microscopy for Single Molecule Tracking
We also developed a microscope that utilizes Interferometric Scattering (iSCAT) to track individual motor domains of dimeric kinesin as they navigate microtubules under physiological ATP concentrations. This iSCAT microscope enables us to image one of the fastest motors known, KIF1A, which takes approximately 200 steps per second when walking on microtubes at a velocity of around 1500 nm/s. We can capture the rapid dynamics of this motor in very high resolution with a frame rate of 100 kHz!
TIRF and iSCAT image of microtubule and Qdot-labeled kinesin motor
TIRF movie shows kinesin motor moving
Our latest addition to the laboratory is the SCATirStorm Microscope, designed specifically for the Cellulase Motility project.
The SCATirStorm Microscope is based on the RM21 framework (Mad City Labs Inc.), featuring two micromirrors positioned at a 45-degree angle beneath the objective lens. These mirrors reflect lasers for Total Internal Reflection (TIR) illumination, allowing for 100% broadband reflection of excitation lasers and 100% broadband transmittance of fluorescence. This unique feature enables us to seamlessly switch between six excitation lasers for multi-color fluorescence imaging without the need to change dichroic mirrors as required in legacy systems.
Cellulase, Qdot-labeled Cel7A(bright objects) , binding to the cellulose surface(black fibers on gray background)
The open-table microscope format grants us significant flexibility. With our current setup, we can utilize iScat, TIRF, Dark Field TIRF, and IRM microscopy techniques. This versatility enables us to visualize a wide range of samples, including large unlabeled polymers, fluorescently tagged molecules, Q-dots, and gold nanoparticles, all with high temporal and spatial resolution.
Relevant Publications:
Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle. K.J. Mickolajczyk, N.C. Deffenbaugh, J. Ortega Arroyo, J. Andrecka, P. Kukura, W.O. Hancock. Proceedings of the National Academy of Sciences. 10.1073: E7186-E7193.
Kinesin Processivity Is Determined by a Kinetic Race from a Vulnerable One-Head-Bound State. K.J. Mickolajczyk and W.O. Hancock. Biophysical Journal. 2017. 112:2615–2623.
High-Resolution Single-Molecule Kinesin Assays at kHz Frame Rates. Mickolajczyk KJ, WO Hancock . Methods Mol Biol 2018; 1805:123-138. doi: 10.1007/978-1-4939-8556-2_7
Interferometric Scattering Microscopy for the Study of Molecular Motors. Andrecka J, Takagi Y, Mickolajczyk KJ, Lippert LG, Sellers JR, Hancock WO. Methods Enzymol 2016; 581:517-539. doi: 10.1016/bs.mie.2016.08.016. Epub 2016 Oct 10.
Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes.Daguan Nong, Zachary K Haviland, Kate Vasquez Kuntz, Ming Tien, Charles T Anderson, William O Hancock