Patent Application
20240288371
The invention relates to the technical field of fluorescence microscopy. More precisely the invention relates to a method for enhancing the photoresistance of a fluorescent species, and a fluorescence microscopy system suitable for implementing said method.
It is concerned with the reduction of the photobleaching of fluorescent labels during the observation of samples by means of fluorescence microscopy.
Fluorescence microscopy is a method for observing a sample, in which the sample emits fluorescence upon being illuminated with an external light source of appropriate wavelength. To obtain the fluorescence, the sample may be coupled to (or labelled, or tagged with) fluorescent species (or fluorophores, or fluorochromes). Fluorophores may for instance be organic molecules, proteins, or nanoparticles able to emit light as a result of an excitation by an external illumination.
As shown on
The long-lived triplet excited state can either
A photobleached state is an irreversible non-fluorescent state of the fluorophore, from which it is not possible to return to the initial fluorescent state neither spontaneously nor by the effect of a light excitation.
For instance, fluorescent species such as organic fluorophores and fluorescent proteins degrade into photoproducts after typically 105 excitation-emission cycles which for a molar absorption coefficient of the fluorophore of 100 000 mol−1·L·cm−1 and an excitation intensity of 1 W·cm−2 at 500 nm leads to the extinction of the fluorophore in about 100 seconds.
Photobleaching results from the gradual extinction of all fluorophores in the sample, by transition from their fluorescent state to photobleached states. The fluorescence signal decay as a function of time may be characterized by measuring a characteristic fluorescence period defined as the characteristic period after which the fluorescence signal is equal to a given fraction of the initial fluorescence signal. For instance, the half-life of a fluorescent species is the characteristic time after which the fluorescence signal is equal to half of the initial fluorescence signal.
Photobleaching concerns all fluorophores and fluorescence imaging techniques and leads to various problems. It limits the duration and/or the time resolution of the experiments, which hinders the proper monitoring of biological dynamics. It limits the number of photons emitted by the fluorophores, which degrades the image quality. It complicates the quantitative analysis of fluorescence variations. In addition, photobleached fluorophore states may be damaging to the sample under study and contribute to phototoxicity.
Several solutions exist to reduce photobleaching. For instance, it is possible to remove oxygen from the sample to avoid the formation of singlet oxygen by interaction of oxygen with the triplet state of the fluorophore. This is usually done by means of enzymes added to the cell culture medium, which reduce oxygen to water, as described in the scientific publications “Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay”, Harada et al., J. Mol. Biol. 1990, 216, 49 and “Oxyrase cell membrane preparations simplify cultivation of anaerobic bacteria”, Thurston et al., Lab. Med. 2000, 31, 509″.
Another existing solution consists in quenching the triplet state of the fluorescent species by means of electron transfer reactions (reduction followed by oxidation, or vice versa). This strategy uses mixtures of reductants and oxidants, such as Trolox or the ROXS system (1 mM ascorbic acid+1 mM methylviologen), a description of which may be found in the scientific publications “Nonblinking and long-lasting single-molecule fluorescence imaging”, Rasnik et al., Nat. Methods 2006, 3, 891” and “A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes”, Vogelsang et al., Angew. Chem. Int. Ed. 2008, 47, 5465”. It requires cell fixation and oxygen removal.
A third strategy can be used to specifically reduce the photobleaching of green fluorescent proteins. Based on the observation that their photobleaching is accelerated in presence of biologically relevant electron acceptors such as flavins, cell culture media were developed which lack riboflavin and pyridoxal. Such culture media are described in the scientific publications “Cell culture medium affects GFP photostability: a solution”, Bogdanov et al., Nat. Methods 2009, 6, 859”, “Anti-fading media for live cell GFP imaging”, Bogdanov et al., Plos One 2012, 7, e53004″ and “Influence of cell growth conditions and medium composition on EGFP photostability in live cells”, Mamontova et al., Bio Techniques 2015, 58, 258″
Hence, existing anti-photobleaching technologies are based on chemically modifying the sample environment. They require time-consuming preparations before imaging and they have side effects on cell physiology which preclude their use for long-term experiments on live cells, as well as the long-term culture of cells in the media used for imaging. Each single product is compatible with only a limited number of fluorophores and biological samples. Moreover, while changing the environment can be a solution for cell cultures or microorganisms, it is not easily applicable to visualize cells in a tissue or in a multicellular organism, where diffusion of the chemicals limits the efficiency of this approach.
One object of the invention is to provide a method for microscopic fluorescence imaging, a method for implementing said method, and a corresponding fluorescence microscopy system which enhance the photoresistance of the used fluorescent protein, i.e. slow down the photobleaching of the fluorescent protein, without chemically altering the sample environment nor requiring any specific sample preparation.
According to an aspect of the invention, it is provided a method for enhancing the photoresistance of a fluorescent protein observed by fluorescence microscopy in a sample containing several molecules of the fluorescent protein according to a fluorescence microscopy technique, the method comprising:
In the context of the invention, the term “absorption band” should be understood as an about 50 nm wavelength interval centered on a peak absorption value, the peak absorption value being a wavelength value for which the absorption reaches a maximum for the considered fluorescent protein. A fluorescent protein may have more than one absorption peak, and therefore more than one absorption band.
In the context of the invention, the intensity should be understood as the irradiance, or power density, that is to say the power received by a surface per unit area.
In the context of the invention, Tmax is the maximum value of the ratio Tγ(λ) for an optimum enhancing wavelength value.
Thanks to the enhancing illumination, the transient state is depleted and the transition of the fluorescent protein into the photobleached state is delayed. Consequently, the photostability, or photoresistance of the fluorescent protein is increased and the photobleaching is slowed down. Reducing the photobleaching rate allows the study of samples for longer periods of time and with a better time resolution, and enhances the quality of the recorded images.
Moreover, the enhancing illumination limits the formation of reactive oxygen species (ROS) which may be harmful for the sample. The phototoxicity is therefore advantageously reduced.
According to an embodiment of the invention, the enhancing wavelength λ+ is an optimum enhancing wavelength chosen so as to maximize the ratio Tγ(λ).
According to an embodiment of the invention, the enhancing illumination is conducted at an optimum enhancing intensity which is the smallest value of the enhancing intensity I+ respecting Tγ′(2I)≤αTγ′(I) where:
According to an embodiment, the illumination period Pi is superior to the half-life period P1/2 of the fluorescent protein when illuminated by the sole exciting illumination, preferably superior to 3 times the half-life period P1/2, and more preferably to 10 times the half-life period P1/2.
According to an embodiment of the invention, the illumination period Pi is superior to 1 second.
According to an embodiment, the exciting intensity Ie is inferior or equal to 1000 W·cm−2 and preferably inferior to 100 W·cm−2.
According to an embodiment, the enhancing intensity I+ respects the relation: 3Ie≤I+≤100 Ie.
According to an embodiment, the enhancing wavelength λ+ is chosen in the near infrared, preferably the enhancing wavelength λ+ is between 700 nm and 1000 nm.
An enhancing wavelength in the near infrared, and preferably between 700 and 1000 nm, advantageously leads to a higher value of the ratio Tγ(λ) and a slower photobleaching of fluorescent proteins. In addition, near infrared illumination is less phototoxic than ultraviolet-visible illumination and has a higher penetration depth in biological samples than ultraviolet-visible light.
According to an embodiment, the exciting and enhancing illuminations are continuous illuminations.
According to an embodiment, the exciting and enhancing illuminations are conducted simultaneously.
According to an embodiment, the illuminations are time-modulated illuminations.
According to an embodiment, the sample comprises living species and is maintained at a non-lethal temperature, and preferably a temperature compatible with the development of the living species. According to an embodiment, the enhancing wavelength is chosen so that the ratio R(λ)=[Q1+2(λ)−Q2(λ)]/Q1 is higher than 1+(Rmax−1)/2 where:
According to another aspect of the invention, it is provided a method for implementing the method according to the invention on a fluorescence microscopy system implementing the fluorescence microscopy technique, the system comprising an illumination system able to deliver at least two light beams in a same illumination region which each wavelength may be chosen among n different illumination wavelengths λk and each intensity is lower or equal to a maximum intensity Imax(λk) reachable by the illumination system for the n illumination wavelengths λk, the method comprising:
According to an embodiment, the method further comprises choosing an optimum enhancing intensity as the smallest value of the enhancing intensity I+ respecting Tγ′(2I)≤αTγ′(I) where:
According to another aspect of the invention, it is provided a fluorescence microscopy system suitable for implementing the methods according to the invention, the system comprising an illumination system able to deliver in a same region at least:
According to an embodiment, the illumination system comprises at least two light sources, one being a tunable light source able to deliver an enhancing illumination with an enhancing wavelength λ+ tunable between at least 700 nm and 1000 nm and an enhancing intensity I+ tunable between at least 20 W·cm−2 and 10 kW·cm−2.
The following description with reference to the accompanying drawings will make it clear what the invention consists of and how it can be achieved. The invention is not limited to the embodiments illustrated in the drawings. Accordingly, it should be understood that where features mentioned in the claims are followed by reference signs, such signs are included solely for the purpose of enhancing the intelligibility of the claims and are in no way limiting on the scope of the claims. In the accompanying drawings:
As explained before, the invention aims at slowing down the photobleaching of a fluorescent protein and reducing the associated phototoxicity when used in fluorescence microscopy, by illuminating the observed sample with both an exciting beam and an enhancing beam.
In order to do so, it may be used a fluorescence microscopy system 1 for microscopic imaging of a sample 2 as shown on
In this embodiment, the system 1 is a wide-field fluorescence microscope comprising the first light source 3 able to deliver the exciting beam 4 having the exciting wavelength selected among n wavelengths λk comprised for example between 380 nm and 650 nm. For example, the first light source is able to deliver an exciting beam at n=10 different wavelengths being: 390 nm or 438 nm or 475 nm or 480 nm or 513 nm or 549 nm or 560 nm or 570 nm or 575 nm or 632 nm. For each wavelength the first light source is able to deliver a light beam having an exciting intensity Ie lower than or equal to a maximum intensity Imax(λk) which is different for each of the n wavelengths λk.
For instance here, the first light source 3 comprises ten light-emitting diodes (LED) 31 one only being shown on the figure. Each LED is configured to deliver a beam 32 collimated by a lens 7 and filtered by an excitation filter 8 in order to form the exciting beam 4. The exciting beam 4 is focused by a lens 33 and directed toward the sample 2 thanks to a first dichroic mirror 9 configured to reflect the exciting beam 4.
In the present example, the second light source 5 is a laser source, here a tunable laser source, able to deliver the enhancing beam having the enhancing wavelength continuously tunable between 700 nm and 1000 nm and an enhancing intensity I+ comprised between 20 W·cm−2 and 10000 W·cm−2.
The enhancing beam 6 is directed toward the sample 2 after a first reflection on a conventional mirror 10, focusing by lens 34 and a second reflection on a second dichroic mirror 11 configured to reflect the enhancing beam 6 and to transmit the exciting beam 4. In this example, the exciting beam 4 passes through the second dichroic mirror 11 after reflection on the first dichroic mirror 9.
The purpose of lenses 33 and 34 is to focus the exciting beam 4 and the enhancing beam 6 in the rear focal plane of the objective 13 so as to obtain parallel beams at the exit of the objective for wide-field illumination of the sample 2. The objective also collects the fluorescence signal emitted by the sample 2 and collimates it into a fluorescence beam 14. An emission filter 15 is configured to filter the fluorescence beam and a second lens 16 is configured to focus the filtered fluorescence beam on a CCD camera 17.
The camera is part of a measurement module 26 configured to measure the quantity Q of fluorescence light emitted by the fluorescent protein, and comprising a processing module configured to normalize the measured signal and to perform operation on the measured signal or on the normalized measured signal, as explained below.
The system further comprises an observation area configured to receive the sample 2 (
The inventors had the merit to demonstrate that it is possible to enhance the photoresistance of the fluorescent protein by illuminating the sample 2 with an excitation beam 4 and an enhancing beam 6. This double illumination with the exciting and the enhancing beams allows longer observation periods than the ones achieved with the sole exciting illumination at a same intensity in both cases.
Thereafter will be disclosed a method for determining the suitable wavelengths and the suitable intensities of the light beams 4, 6, in which the above-described system 1 for fluorescence microscopy is used for observing the sample 2 being labelled with a chosen fluorescent protein. The sample 2 may be labelled using any labelling method known in the art, and the man skilled in the art knows how to choose the appropriate method according to the considered application and the used fluorescent protein.
As shown on
The sample 2 is illuminated with the exciting light beam 4 at an exciting wavelength de selected among the n different illumination wavelengths λk so as to be in an absorption band of the fluorescent protein, in order to trigger a fluorescence emission by the fluorescent protein. For instance, the exciting wavelength λe is selected among the n wavelengths λk to be the closest from the absorption peak of the fluorescent protein. Those skilled in the art know how to choose the appropriate value for the exciting wavelength as many databases disclose this information which is also given by the providers of fluorescent proteins.
The exciting wavelength de value is preferably selected within a range of about 50 nm centered around the maximum absorption value.
The exciting intensity Ie is set according to the fluorescence microscopy technique (epifluorescence microscopy, confocal microscopy, etc.) and lower than 10 kW/cm2. Those skilled in the art know how to choose the exciting intensity. For instance, in wide-field microscopy the exciting intensity Ie is typically less than 10 W·cm−2, while in laser scanning confocal microscopy the exciting intensity Ie is typically higher than 1 kW·cm−2.
As a first example of implementation the fluorophore is the Enhanced Green Fluorescent Protein (EGFP) embedded in a polyacrylamide gel. The absorption peak of the EGFP is known as being 488 nm, therefore the exciting wavelength is selected at 480 nm which is the wavelength nearest to the absorption peak among those available in the system 1.
As shown in
The implementation is therefore conducted using wide field illumination with an exciting intensity Ie of 32 W·cm−2.
The exciting wavelength λe and the exciting intensity Ie being set, the best setting for the enhancing beam 6 has to be determined. The enhancing beam 6 should be configured to be absorbed by the fluorescent protein so as to trigger a transition of the fluorescent protein from the transient state back into the initial state. It is advantageous to first configure the enhancing wavelength λ+ namely the wavelength within the range of available wavelengths which has the enhancing effect demonstrated by the inventors. Once this enhancing wavelength is identified, it may be advantageous to optimise the enhancing intensity I+ in order to limit the light received by the sample and hence the possible side effects of the enhancing illumination. At first for determining the enhancing wavelength, an arbitrary predetermined enhancing intensity value may be selected during the configuration of the enhancing wavelength, and preferably a low enhancing intensity value. Here, the arbitrary enhancing intensity value is set to 20 W·cm−2.
Thus, in a first step of the method, the instant fluorescence emitted by the fluorescent protein as a function of time during the illumination is measured in presence and absence of the enhancing illumination and for different values of the enhancing wavelength λ+.
The enhancing wavelength λ+ value should be selected as enhancing the photoresistance of the fluorescent protein, that is to say it should be selected so as to slow down the decay of the fluorescence triggered by the exciting illumination only, in presence of the enhancing illumination
Thus, a comparison is performed between
This comparison is quantified as the ratio: Tγ(λ)=P12,γ(λ)/P1,γ. The ratio Tγ is studied for all the available values of the enhancing wavelength λ+.
To this end, several measurements of the instant fluorescence emitted by the fluorescent protein as a function of time with both the exciting beam 4 and the enhancing beam 6, or with only one of the beams 4, 6, are performed. Since the photobleached state is irreversible, two distinct measurements may not be performed on the same location of the sample 2.
For instance, it is retained an optimum enhancing wavelength λ+ value for which the slowing down of the photobleaching is at least half of its maximum value, i.e. an enhancing wavelength value for which Tγ(λ+)>1+(Tmax−1)/2, where Tmax is the maximum value of Tγ for the predetermined enhancing intensity value.
More preferably, it is retained the optimum enhancing wavelength value for which Tγ(λ+)=Tmax. Here, as apparent on
Once the optimum enhancing wavelength is determined and set, it is advantageous to determine an optimum enhancing intensity I+ in relation with said optimum enhancing wavelength λ+ value previously determined.
In order to do so, the value of the ratio Tγ′(I)=P12,γ′(I)/P1,γ is studied for all the available values of the enhancing intensity I+ (which depend on the configuration of the system). Preferably, the enhancing intensity is limited to values higher than the exciting intensity Ie. For instance, the enhancing intensity is limited to values respecting 3Ie≤I+≤100Ie.
It may be observed that the value of the ratio Tγ′(I) increases with the enhancing intensity I+. In order to limit sample illumination and any side effect that might arise from it, choosing the maximum available value for the enhancing intensity I+ may however not always be the best choice.
Since the value of the ratio Tγ′(I) forms a plateau above a certain value of the enhancing intensity, further increasing the enhancing intensity is not advantageous. It is preferable to choose a minimum enhancing intensity value leading to a ratio Tγ′(I) belonging to the stage.
To this end, the optimum enhancing intensity I+ is chosen so as to respect Tγ′(2I+)≤αTγ′(I+), where
Here, the selected enhancing intensity is equal to 2 KW·cm−2 and Tγ′(I)=3.5.
Once the enhancing wavelength λ+ value and the enhancing intensity I+ value have been determined for a specific fluorescent protein (here, EGFP), they may be used in order to enhance the photoresistance of this fluorescent protein, for instance in a microscopy experiment that involves a sample labelled with this fluorescent protein or for any type of experimentation, observation or measurement which may be conducted with this specific fluorescent protein.
The effect of the enhancing illumination demonstrated by the inventors is evidenced by the study of the ratio Tγ as disclosed before.
On
It is apparent on
Those values of the half-life ratio T1/2(λ)=P12,1/2/P1,1/2 are a consequence of an increase in the fluorophore photoresistance and a slowed down photobleaching due to the double illumination of the fluorophore. It is therefore possible to acquire fluorescence data over a longer period.
The above-described embodiment applies to the determination of the enhancing wavelength \+ and the enhancing intensity I+ of the EGFP. However, the determination method may apply to any other fluorophore.
Moreover, in the above-described example, the fluorophore was embedded in a polyacrylamide gel. However, the invention is not limited to fluorescent proteins embedded in polyacrylamide gels and the advantageous effects provided by the invention may be obtained with fluorescent proteins coupled to any suitable support, for instance biological samples such as bacteria or eukaryotic cells.
As a demonstration of the relevance of the invention for enhancing the photoresistance of fluorophores in biological samples,
The
For this implementation, the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 32 W·cm−2, the enhancing wavelength λ+ is equal to 900 nm and the enhancing intensity I+ is equal to 2 KW·cm−2.
It is apparent on
On each micrograph, the bright spots are Hela cells 23 labelled with cytoplasmic EGFP and emitting fluorescence. All the cells are illuminated with the exciting beam (wide field illumination) and the cells located in the second illuminated area 20 (represented by a dotted circle) are illuminated by both the exciting beam 4 and the enhancing beam 6.
It is clear on
In this example, the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 32 W·cm−2, the enhancing wavelength λ+ is equal to 900 nm and the enhancing intensity I+ is equal to 125 W·cm−2.
It is apparent on
In this example, the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 32 W·cm−2, the enhancing wavelength λ+ is equal to 900 nm and the enhancing intensity I+ is equal to 125 W·cm−2.
It is apparent on
The invention is not limited to implementations with the Enhanced Green Fluorescent Protein (EGFP) but may be implemented with other green fluorescent proteins.
For instance, in an embodiment with live Escherichia coli bacteria labelled with green fluorescent proteins in which the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 32 W·cm−2, the enhancing wavelength λ+ is equal to 900 nm and the enhancing intensity I+ is equal to 125 W·cm−2,
The invention is not limited to green fluorescent proteins but may be implemented with fluorescent proteins of other colours, for instance red or yellow.
For instance, in an embodiment where the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 37 W·cm−2, the enhancing intensity I+ is equal to 125 W·cm−2, and the enhancing wavelength is equal to 900 nm, the fluorescent protein being Venus in live Escherichia coli bacteria, the half-lives ratio T1/2 is equal to 1.8.
In another embodiment where the exciting wavelength λe is equal to 480 nm, the exciting intensity Ie is equal to 37 W·cm−2, the enhancing intensity I+ is equal to 125 W·cm−2, and the enhancing wavelength is equal to 900 nm, the fluorescent protein being the EYFP (Enhanced Yellow Fluorescent Protein) in live Escherichia coli bacteria, the half-lives ratio T1/2 is equal to 1.8.
In other embodiments with red fluorescent proteins in live Escherichia coli bacteria where the exciting wavelength λe is equal to 560 nm, the exciting intensity Ie is equal to 5.8 W·cm−2, the enhancing wavelength λ+ is equal to 725 nm and the enhancing intensity I+ is equal to 250 W·cm−2,
As mentioned above, the method according to the invention not only has a beneficial effect on photobleaching, but also reduces phototoxicity.
It appears that the development of the bacteria is slowed down when the sample is illuminated only by the exciting illumination and that the double illumination makes it possible to obtain a normal development of the bacteria, i.e. a development identical to that which occurs in the absence of any illumination.
Although the previously described embodiments relate to a continuous illumination, the invention may be performed with different kinds of illumination, for instance a scanning illumination, a pulsed illumination, or a time-modulated illumination. More precisely, the exciting and enhancing illuminations may be of different kind and may be carried out simultaneously or not. In embodiments where at least one illumination is a scanning illumination, the corresponding illuminated area should be understood as an area covered by said scanning illumination.
Although several examples previously described relate to a characteristic period being the half-life T1/2, the invention may be performed with different characteristic periods, for instance the period T0,1 after which the fluorescent signal has lost 10% of its value, or the period T0,9 after which the fluorescent signal has lost 90% of its value.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21305834.0 | Jun 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/066617 | 6/17/2022 | WO |
