Prism-Type TIRF (2x two-color, 2x four-color)

The Ha-Myong Lab has two each of two-color and four-color prism-type TIRF microscopes. All four microscopes have maximum fields of view of very approximately 200 by 200 microns. Notably, this field of view is decreased when additional color channels are used, as the channels are aligned side-by-side on the 512 by 512 pixel image sensor. The two-color TIRFs are functionally identical and are each equipped with a 532 nm laser and a 640 nm laser. One of the four-color TIRF microscopes is presently under construction, with its camera and associated optics unfinished. This microscope possesses lasers of 488 nm, 532 nm, 647 nm, and 730 nm, while the older already-constructed four-color TIRF possesses a 543 nm laser instead of a 532 nm one. Except for the constructed four-color microscope, the prism-type TIRF microscopes and their optical components are not isolated.

Objective-Type TIRF Live Cell

The objective-type TIRF microscope is preferential to the prism-type TIRF microscopes for live cell TIRF imaging, as the evanescent field (the electromagnetic field responsible for TIRF florescence) is generated from the direction of the coverslip, where cells are grown. However, objective-type TIRFs ultimately attain lower signal-to-noise ratio than prism-type, for which laser light does not enter the objective. The Ha-Myong Lab objective-type TIRF microscope is a four-color microscope capable of applying light at wavelengths of 488 nm, 543 nm, 561 nm, and 640 nm to samples. A 750 nm laser power supply is also available. As with the prism-type microscopes, the maximum field of view of the objective-type TIRF microscope is around 200 microns by 200 microns. This microscope is isolated.

Wide Field Live Cell

The wide field microscope is a simple-to-use commercial microscope offering around a 130 by 130 micron field of view with its 100x objective and a 200 by 200 micron field of view with its 60x objective. The microscope uses six light sources (LEDs or luminescent pipes) and filters to selectively excite fluorophores. The six available excitation wavelengths are 390 nm, 438 nm, 485 nm, 560 nm, 650 nm, and 740 nm. The microscope is capable of FRAP, for which it employs a focused 405 nm laser. The nosepiece of the microscope is somewhat finnicky, with stickiness observed around positions 5 and 6, however this has no impact on its experimental capabilities. The microscope is isolated.

Light sheet/SIM Live Cell Super-Resolution

This microscope possesses both light sheet and Structured Illumination Microscope (SIM) capabilities. Loading samples is somewhat inconvenient. The microscope is equipped with lasers providing excitation wavelengths of 405 nm, 488 nm, 560 nm, and 650 nm. The field of view of the microscope for lattice light sheet microscopy is 100 by 100 by 15 microns and is limited by the travel range of the scanning piezo stage. The length of the light sheet is 30 microns. The light sheet is very similar to the wide field microscope, except that use of the light sheet allows the microscope to selectively illuminate only a single z axis layer at a time. When functioning as an SIM, a type of super-resolution microscope that illuminates the sample with a wave pattern and shifts the pattern to generate an image, the microscope can improve resolution to roughly 70-80 nm.

STED Live Cell Super-Resolution

The Ha-Myong Lab STED (stimulated emission depletion microscopy) is a scanning microscope that can attain around 50-60 nm resolution. The microscope possesses excitation lasers with wavelengths of 594 nm and 650 nm, as well as a depletion laser of wavelength 775 nm. The microscope could be used for live cell imaging; however, it is not presently optimized for it. Optimizing the microscope for live cell imaging would require a faster scanner and fluorescent tags with high brightness and high depletion efficiency. Without these improvements, the STED microscope could struggle to capture fast behaviors in cells and would risk killing sample cells through phototoxicity. The microscope has a field of view of 20 by 20 microns and is not presently isolated.

STORM/DNA-PAINT Super-Resolution

This microscope can run STORM or DNA-PAINT experiments and achieves the highest resolution of any Ha-Myong Lab fluorescence microscope. The microscope has four lasers of wavelengths 405 nm, 488 nm, 568 nm, and 642 nm. The microscope applies laser light to the sample through the objective, allowing it to be used for objective-type TIRF. When used for STORM the microscope attains resolutions in the tens of nanometers (on the order of 20-30 nm), while resolutions for DNA-PAINT are around 10 nm. Maximal field of view is about 150 by 150 microns. The microscope’s performance is degraded by a gradual drifting in the sample position over the course of imaging, which requires registration to correct during processing. The microscope is not isolated, which also reduces resolution. Notably, data collection for DNA-PAINT or STORM is a long process, as both techniques rely on fluorophore blinking. When collecting DNA-PAINT or STORM data, the microscope must collect thousands of images to identify the center point of each fluorophore’s Airy Disk. Fortunately, an autofocus is available to keep the microscope focused over this period, however the autofocus is old and not well documented.

Optical Tweezers

The Ha-Myong Lab uses a Lumicks C-Trap for optical tweezer experiments. This instrument uses mirror-steered laser beams to trap beads and can deploy up to four trapping beams simultaneously. The C-Trap incorporates a confocal microscope for fluorescence experiments. The microscope scans along an axis parallel to the trapping beams (and thus perpendicular to samples suspended between them), typically requiring about 80 ms to generate each frame with a field of view of 50 by 35 microns. The C-Trap is equipped with excitation lasers of wavelength 488 nm, 561 nm, and 638 nm for confocal fluorescence microscopy. The C-Trap also incorporates a five-channel automated microfluidics system to load samples. Based on manufacturer’s specifications, the force resolution of the optical tweezers is <0.1 pN at 100 Hz, with force stability of <0.3 pN over two minutes for 1 micron beads at or above .35 pN/nm trap stiffness. The minimum incremental step size is 0.2 nm, with a 50 by 50 by 9 micron field of movement. The manufacturer claims the confocal microscope’s scanning speed can reach 100 Hz. More detailed specifications are available in the product brochure.

Atomic Force Microscopy (AFM)

The AFM microscope is the only Ha-Myong Lab microscope not to rely on fluorescence. AFM functions by placing a sharp tip attached to a cantilever into contact with a sample and using piezoelectric transformers to move the sample across the tip. A laser is employed to detect the movement of the tip as it passes over DNA, proteins, or other obstacles. Use of AFM requires the imaging target to reside on a scanned surface, which can be charged to attract biological molecules. The AFM’s resolution is determined by scanning speed and by the sharpness of the instrument’s tip. Under typical experimental conditions, the Ha-Myong Lab instrument attains resolutions of 4 nm to tens of nm. Generally, each frame is scanned over the course of around 50-100 ms. The field of view is generally around 4 by 4 microns; this area may be limited (reducing the area over which the tip must scan) to improve framerate or resolution.