Written by Teng-Leong Chew
Photophysics of photobleaching
A molecule will emit fluorescent light when its electron, falling from the excited state to the ground state, releases most of the energy that it absorbs during excitation. Upon returning to the ground state, the electron is ready to be excited again for another productive cycle. However, these cycles will not continue forever. In some cases, the electron does not return to the ground, but is instead trapped in an esoteric photophysical state called the triplet state. The presence of oxygen radicals, for example, in the microenvironment can often siphon the triplet state electron away from the productive cycle of fluorescence emission. This process of ‘robbing’ the electron permanently away from a fluorescent molecule is the essence of photobleaching. This is summarized in the Jablonski diagram below.
Quantum yield and photon budget
Photobleaching thus defines the permanent loss of fluorescence due to photo-induced chemical changes. On the other hand, quantum yield describes the fractions of photons absorbed that will be re-emitted by the fluorophore. Every fluorescent molecule has its own photobleaching rate and quantum yield. There are several fantastic online references one should consult to find out these important numbers for the fluorophore of interest.
Interactive Visualization of Fluorescent Protein Properties: http://www.fpvis.org/
Fluorescent Protein Database: https://www.fpbase.org/
Together, quantum yield and photobleaching rate contribute to a very important factor in light microscopy: Photon budget. Photon budget is the number of detectable photons that a chosen fluorophore can contribute to the experiment at a given period of time. In practical terms, it sets a limit to how many image acquisitions can be performed on a sample. The quantum yield of a chosen fluorophore will determine, to a large degree, the minimum exposure time (milliseconds/frame) that can be used, and how much excitation light is necessary during the given acquisition time to obtain an image with acceptable signal-to-noise ratio (SNR). Likewise, photobleaching rate will limit how many frames one can acquire – at such high speed wherein the replenishing effect of newly synthesized fluorescent molecules is negligible – before fluorescence from the sample becomes undetectable.
Planning your experiment with photon budget in mind
Many prospective applicants to the Advanced Imaging Center (AIC) are attracted by the high-speed microscopes available at the center and during technical consultation are understandably concerned about how fast the microscopes could acquire 3D volumetric image stacks. However, the limiting factor is usually not the microscope; it is the photon budget. The AIC review committees pays close attention to the fluorophore choice in each proposal we receive because photon budget is one of the most commonly neglected factors that would significantly impact the feasibility (let alone the success) of an experiment – and hence the outcome of one’s proposal to the AIC.
During technical consultation, we are frequently asked if the AIC instruments can operate fast enough to capture 1-2 volumes per second for a prolonged period of imaging duration. While that speed is certainly within the operating parameters of some of the AIC instruments, the factors that would limit the feasibility of this project is the photon budget and the photostability of the biospecimens.
The two sets of slide bars below illustrate the limiting effects of photon budget. The lengths of the slide bars represent the relative photon budgets of two red-emitting fluorophores, mCherry and mRuby2. The AIC discourages prospective applicants from tagging their target proteins with mCherry due to its poor photon budget. In fact, in most cases, the choice of fluorophores with lower photon budgets, such as mCherry, RFP, CFP, BFP, YFP may lead to technical rejection of a proposal at the Tier 1 review. The reason is self-evident below. With limited photon budget, one now has a much narrower range of maneuvering room with mCherry and is immediately forced to make a choice between imaging speed and number of acquired frames.
Explore how these properties relate further with interactive slide bars bellow. The length of each slide bar is relates to the quantum yield of the fluorophore.
This is also another reason why a technical consultation with the AIC is so important.
The AIC recommends the following genetically encoded fluorophores for live-cell imaging
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