The resolution of a regular fluorescence microscope image is limited by diffraction to approximately half the wavelength of the emitted light. To separate fluorescently labeled structures that are closer together, a solution is needed to overcome Abbe’s diffraction limit. The most common super-resolution methods for imaging beyond the diffraction limit are localization microscopy, SIM (structured illumination) and STED (Stimulated Emission Depletion Microscope).
Ground State Depletion followed by individual molecule return (GSDIM) is a localization microscopy technique. In general, localization microscopy does not look at an ensemble of simultaneously emitting fluorophores, but at clearly separated, individual fluorophores which can be localized with nanometer precision. Over time, the position of each fluorophore labeling a biological structure is determined and the image is re-constructed in silico based on the position information of each fluorophore. The challenge is to effectively singularize an ensemble of fluorophores to allow single molecule detection.
GSDIM – the molecular basics
The crucial step in GSDIM is to temporarily switch the majority of fluorophores off to allow the precise localization of single fluorophores. To this end, excitation light of high intensity is used in such a way that almost all fluorophores in the sample are turned dark, leaving only single, well-separated fluorophores emitting fluorescence, which is a prerequisite for nanometer precision localization.
Fig. 1 (right): Schematic representation of the GSDIM method based on a simplified Jablonski diagram. Delocalized π electrons in fluorophores can be, for instance, in the ground state S0>, in the excited state S1 (both so-called ON states) or in a triplet or radical dark state (both OFF states). When fluorescent light is emitted, electrons circulate between the ground and the excited state. Unlike these ON states, fluorophores in the OFF state are not able to emit light. These OFF states are usually of long lifetime, but they are difficult to attain, as an inter-system crossing is required. By setting the right ambient conditions in the embedding medium and through the clever choice of standard fluorophores for immunofluorescence, it is possible to reversibly switch off fluorophores by exciting them with an extreme light intensity. When enough molecules are in the OFF state, it is possible to detect individual molecules in the sample.
Transitions between molecular states
As soon as a fluorescent sample is excited by (laser) light, electrons are catapulted by photons into the 1st excited state. On returning from the 1st state into the ground state, light of slightly lower energy (red-shifted) is emitted. Higher laser power increases the number of photons that can hit an electron in the ground state and transfer the electron into the excited state. As a result, more electrons in the sample cycle between the ground and excited state in a given time. The number of emitted photons increases and a brighter sample is visible.
A further increase of the excitation light (e.g. from a high power laser source) can lead to a dimmer sample due to reduced fluorescence emission. At the beginning of this process, the increase in excitation light leads to a higher number of electrons cycling between the ground and the 1st excited state. From the excited state a certain percentage of electrons enter the off state. The only accessible sub-state from the 1st excited state is the triplet state. Entering the triplet state requires a spin-flip of the electron. A spin-flip has a very low probability of occurrence and is therefore a very rare event. In consequence the off state is practically unpopulated at low light exposure of the sample.
Off states have a long lifetime (in the range of μs to s) and fluorophores with electrons in the off state cannot participate in fluorescence emission of the sample any more. Over time more and more electrons end up in the off state and the sample appears dimmer although the overall number of fluorophores has not changed. Just the number of “active” or “accessible” fluorophores has been altered. “Pumping” electrons in the off state can be seen as a reversible switch. Fluorophores are turned off for the time the electron resides in the off state. When the electron returns to the ground state, the fluorophore is rendered “active” and can again emit fluorescent light. All transitions between molecular states of electrons are probability-governed processes and therefore the exact occurrence of a transition cannot be predicted for a given electron.
Localization up to nanometer precision
For super-resolution imaging the laser power is kept at a high level until only a few fluorophores in the field of view emit photons. The "active" fluorophores can cycle up to a few thousand times between the ground and 1st excited state, creating a “burst” of photons before the electron returns to the off state. The photon “burst” is recorded with a high-sensitive EMCCD camera. Furthermore, only a few electrons populate the ground state for a short time, leading to the situation of "ground state depletion with individual molecule return" (GSDIM). In general, fluorophores emitting photon bursts are spatially well separated, which allows the allocation of all photons in a certain region to a single molecule. In this situation the center of the recorded, diffraction-limited signal is the same as the position of the fluorophore. The position of the fluorophore can be calculated up to nanometer precision.
Fig. 3 (right): Widefield versus GSDIM. Golgi membrane protein Giantin and Golgi matrix protein GM130, immunofluorescence staining with Alexa Fluor 647 and Alexa Fluor 488, respectively. Courtesy of Dr. Yasushi Okada, Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Japan.
Why organic fluorophores are better
The final resolution of an super-resolution image depends on a) the precision of the localization of each individual fluorophore (localization precision) and b) the minimal distance between individual fluorophores marking the structure of interest (staining density). The staining density can be influenced e.g. by higher antibody concentrations used during immunostaining. The localization precision is dependent on the number of photons collected from an individual fluorophore and can be approximated by the following formula:
Δr: Diffraction limit of the microscope/objective
N: Number of photons emitted by an individually localized fluorophore
Δsms: Localization precision of the individual fluorophore
Therefore, to achieve a localization precision of 10 nm, 400 photons have to be collected from the fluorophore with a microscope delivering a diffraction limited resolution of 200 nm. The number of photons emitted is in the first instance a property of the fluorophore in combination with the embedding medium. As a rule of thumb, organic fluorophores display higher brightness and photostability than fluorescent proteins. In consequence the number of photons delivered by these dyes is much higher and the localization precision significantly improved. Finally, a better super-resolution image can be obtained with “stronger” dyes like organic fluorophores due to the improved localization precision!