Ny forskningsrapport från Nobelpristagare Stefan W. Hell

Nobelpristagaren Stefan W. Hell m kollegor har publicerat en ny studie som handlar om förbättrad teknik att avbilda celler in i minsta detalj. Studien är alltför tekniskt komplicerad för en lekman att förstå, så jag ger mig inte in på att försöka förklara i detalj vad det handlar om.

Det jag kan kommentera är att Hell använt sin HoloMonitor i kombination med andra mikroskop,b.l.a med STED-mikroskopet han fick sitt Nobelpris för. Så studiens resultat har alltså HoloMonitor medverkat till vilket får anses vara en kvalitetsstämpel (signerad av Mr Hell himself).

N-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins

Abstract

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their O-unprotected forms. Herein, we report rationally designed N-cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from N-cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells.

Introduction

One of the most important advantages of small molecule fluorescent probes over genetically encoded fluorescent proteins is their superior photostability,which becomes essential under the demanding conditions of super-resolution microscopy (nanoscopy).Recent advances in fluorescence nanoscopy have prompted the rational design of small molecule fluorescent labels^. Achieving live-cell compatibility with high target specificity, and the ability to control the net charge of a fluorescent probe, while maintaining high brightness, chemical stability and low photoreactivity of the fluorophore remain considerable challenges.

Fluorescein (resorcinolphthalein) is a green-emitting (λ_exc = 498 nm, λ_em = 517 nm, fluorescence quantum yield Φ = 0.90 in 0.1 M NaOH)⁷ fluorescent dye from the phthalein family of triarylmethane dyes, first reported by von Baeyer in 1871. While its pK_a value of 6.3 makes it brightly fluorescent at cytosolic pH = ∼7.2,the anionic form does not cross the cell membranes of mammalian and plant cells. On the contrary, its non-fluorescent O-acyl esters,in particular fluorescein diacetate, enter living cells and are hydrolyzed by cell esterases into free fluorescein. Employing self-labelling protein tags such as HaloTag,CLIP-tag and SNAP-tag,fluorescein ester-derived cell-permeant and fluorogenic live-cell probes targeting specific fusion proteins have been developed. Fluorescein itself, however, undergoes comparatively rapid photobleaching with a quantum yield of Φ_bl = 3 × 10⁻⁵ in water.Its 2′,7′-difluoro derivative (Oregon Green) is somewhat more photostable and has found wider use in fluorescence microscopy. Besides O-acylation, photocleavable and enzymatically cleavable O-protecting groups have been used to render fluorescein and Oregon Green dyes and their derived probes cell-permeant. Replacing the oxygen bridge (X = O) in the xanthene core of fluorescein affords its analogues (thiofluorescein X = S), carbofluorescein (X = CMe₂) and Si-fluorescein (X = SiMe₂)) with lower LUMO energies and red shifts of both fluorescence excitation and emission maxima . 

Their photostabilities, however, have not been systematically studied.

Verifiering av användande av HoloMonitor hittas i själva studien, men mer fyllig information finns i Supplementary Information.

Biocompatibility and cellular imaging

The cell morphology and proliferation rates of living U-2 OS cells, monitored by means of holographic imaging cytometry in the presence of 5 μM of 5a, 6a or various CR1–CR3 derivatives in the culture medium, were unaffected over at least 48 h (Fig. 5B–D and S14).

Fig. 5 (A) CellTiter-Blue cell viability assay showing no toxicity for 5a, 6a and TMR in comparison with DMSO vehicle control (U-2 OS cells, over 24 h). (B–D) U-2 OS cell proliferation as followed by holographic time-lapse imaging cytometry. (B) Cell division frequency over 48 h in presence of 5 μM dye. (C) Unaltered cell count and (D) visualization of a normal cell division in the presence of 5 μM CR1-Halo. Scale bar: 50 μm. Statistical significance: ns – no significant difference to control, *p < 0.05, **p < 0.005,

 

Figure S14. Assessment of cytotoxicity. (A) Schematic depiction of digital holographic microscopy. A cell delays the phase of the sample beam due to an increased refractive index as compared to its surrounding medium. Its phase image is computed from the hologram created by interference of the phase-shifted sample beam and the reference beam. (B) Normalized U-2 OS cell count and (C) U-2 OS cell cycle length as followed by holographic time-lapse imaging cytometry over 48 h in presence of 5 µM compound. (D) The CellTiter-Blue cell viability assay is based on the ability of viable cells to reduce the non-fluorescent redox indicator resazurin to the fluorescent dye resorufin. As non-viable cells rapidly lose this metabolic capacity, the fluorescent signal is directly proportional to the number of living cells. (E) CellTiter-Blue cell viability assay with 1% Triton X-100 (TX100) as positive control (U-2 OS cells, over 24 h, all compounds at 5 µM). Statistical significance: ns – no significant difference to control, *p<0.05, **p<0.005, ***p<0.0005; detailed information is provided in Table S3 and S4.

Holographic time-lapse imaging 

 

Potential cytotoxicity of the dye cores and the HaloTag/SNAP-tag ligands of the Ncyanorhodamines, TMR and spectrally similar Janelia Fluor (JF) dyes was assessed by monitoring U-2 OS cell proliferation via holographic time-lapse imaging and by analyzing the frequency of cell division and the cell cycle length. U-2 OS cells were seeded in lumox 96-multiwell plates (94.6120.096, Sarstedt; 100 µL/well, 104 cells/mL) 24 h prior to the experiment and kept at 37 °C in humidified air with 5% CO2. Stock solutions of the the Ncyanorhodamine fluorophores and the derived HaloTag/SNAP-tag ligands as well as spectrally similar rhodamine dyes were prepared in DMSO (5 mM). 

 

Imaging was performed with a HoloMonitor® M4 cytometer (PHI AB) for a total period of 48 h with 30 min between image captures at 37 °C in humidified air with 5% CO2. Cell proliferation was followed in absence and in presence of 5 µM dilutions of CR1-Halo, CR1-BG, CR2- Halo, CR3-Halo, 5a, 6a, N,N-dimethylrhodol (10), HaloTag TMR Ligand (G8252, Promega), JF549 HaloTag Ligand (GA1110, Promega), JF585 HaloTag Ligand (CS315105, Promega), TMR and JF549 in DMEM. 

Data analysis comprised cell segmentation, tracking of cells, identification of dividing cells, and cell counting with the AppSuite 3.5 (PHI AB) software.

 

Min kommentar

Finge man önska skulle PHI jobba som f-n på att få Mr Hells godkännande att på varje HoloMonitor ha tryckt : Approved by Nobel Prize winner Stefan W. Hell.

Eller ha med samma slogan i all marknadsföring. Det skulle göra under för försäljningen. 😎

                                              Mvh the99

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