Imaging the Genome
a given focal position, and scanning the objective to image multiple focal planes allows for imaging vol- umes greater than 30 µm thick. Te underlying principle of SML relies on the abil-
ity to isolate a single molecule in a diffraction-limited region at a given time. Tere are two main methods for obtaining optical control over fluorophores that enables localization of molecules. Fluorescent proteins that switch from an “off” state to an “on” state with ultraviolet light (photo-activatable proteins [17–19]) or ones that shiſt emission spectra (photo-convertible [20]) can be used. A more widely used method uses organic dyes that, through a series of reversible light- driven reduction reactions, bind a side-group to the fluorophore, rendering it non-fluorescent [21–23]. Te reversibility of the reaction enables a blinking behav- ior that allows a single fluorophore the opportunity to be localized multiple times during an experiment. While routinely used in super-resolution microscopy methods, such as (d)STORM, STORM, and GSDIM, the lack of available fluorophores with appropriate photoswitching properties makes imaging many tar- gets in a single experiment quite challenging [24]. An alternative approach to SMLM using organic
Figure 2: Overview of SMLM. Schematic overview of single-molecule localization micros- copy. Top left: Cartoon showing blurring of feature by conventional wide-field imaging. Right column: Fluorescent molecules are photochemically switched to a dark state; a sparse subset of molecules is switched back to a bright state, and their centers are localized computationally. This process of switching to a dark state, activating, and being localized is repeated many times to obtain the final super-resolution image. Bottom left: All localization molecules are compiled into one final super-resolution image with well-defined features compared to widefield.
dyes without relying on photoswitching to gener- ate blinking is a technique called Points Accumula- tion In Nanoscale Topography (PAINT) [25]. With this method and its derivatives, blinking is gener- ated by the transient binding of a fluorophore to a target. When the fluorophore is bound, it appears in focus on the imaging camera, and the position can
Figure 3: Multiplexing with DNA-PAINT. (A) DNA-PAINT schematic. The process begins with DNA-labeled antibodies bound to the sample (A and B), shown here as primary labeled antibodies for simplicity. A solution containing oligonucleotides complementary to antibody A bearing fluorophores (A*) is applied to the sample during imaging. A* transiently binds and unbinds to A during image acquisition. A* probes are washed from the sample, so it no longer contains any fluorescent- emitting molecules. Antibody B is then labeled and imaged in the same manner using a different set of complementary oligonucleotides (B*). (B) DNA-PAINT images of microtubules (green) and clathrin (magenta), imaged sequentially.
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