Hs68 fibroblasts were acclimated to advance a connected workflow for investigating telomeric appearance and samples were able according to the FISH agreement declared in the methods section. Wide-field microscopy was acclimated for angel accretion and a breeze blueprint summarizing the procedures declared beneath is apparent in Fig. 1 (Supplementary Fig. 1 for abundant protocol). Wide-field microscopy is sometimes advised inferior to laser scanning confocal microscopy apropos resolution, yet camera-based systems are added sensitive, cost-effective, and can be added time able back imaging in 3D28. Furthermore, wide-field microscopy, back accumulated with accepted able agenda deconvolution methods, yields optical sections commensurable to the optical sectioning inherent in confocal techniques. Another advantage is that the camera technologies, such as authentic commutual metal–oxide–semiconductor (sCMOS) cameras, acquiesce advisers to access beyond fields-of-view, appropriately abbreviation beginning time at the wide-field microscope29.
Flow blueprint summarizing procedures declared in this study. For a abundant and stepwise protocol, see Supplementary Fig. 1.
To ensure bendability and reproducibility, samples were beheld application the aforementioned microscope settings, including beam intensity, acknowledgment time and footfall admeasurement of Z-stacks. Back accepting images, it is important to admit the acuteness ethics that accord to the cardinal of emitted photons detected by photosites on the camera. The acuteness ethics are bidding over a ambit that is authentic by the bit-depth (N) of the camera to call the bulk of grayscale that can be represented in the final angel (2N). For example, a 12-bit camera will apprehend out pixel acuteness ethics alignment from 0 to 4095 (=212). Accepted convenance for immunofluorescence anchored imaging is to ample 50–75% of the bit abyss of the camera. The pixel acuteness administration can be gauged by the acuteness histogram in the imaging software and is abased on the acknowledgment time and acuteness settings. Pixel assimilation was abhorred back it leads to baloney of intensities as it does not reflect the absolute cardinal of photons hitting the photosites in the camera. Back recording volumetric data, the footfall admeasurement of Z-stacks should abduction all fluorescence arresting in a 3D amplitude and this can be accomplished by imaging at the Nyquist bulk in both crabbed (X, Y) and axial (Z) directions30. Furthermore, to ensure the absolute sample is imaged, the top and basal banned of the Z-stacks should be set to almanac dim and out-of-focus fluorescence. If these altitude are set incorrectly, consecutive angel pre-processing steps, such as deconvolution, will be decumbent to artefacts31. During our imaging, a footfall admeasurement of 70 nm was acclimated for X, Y and 100 nm for Z.
To accurately calculation telomeres in interphase, PNA staining of a metaphase advance acquired from the beef of absorption was performed18. This is an important footfall to ensure that the PNA delving binds accurately at the chromosome ends beneath the performed beginning altitude and to affirm the cardinal of telomeres per cell. This metaphase appraisal will additionally accede if any chromosomes arise to accept telomere-free ends or end-to-end fusions32, which would abate the calculation of predicted telomeres in interphase cells. Furthermore, both sister telomeres in a metaphase advance should accept the aforementioned staining acuteness back the majority of sister telomere pairs accept according lengths33. In our experiments, back assuming a metaphase advance of Hs68 fibroblasts, 46 chromosomes were stained, and anniversary chromatid-end was telomere absolute (Fig. 2a, b). This reconfirms that our accustomed diploid fibroblast beef accommodate 92 telomeres while in the G0 and G1 phases of the corpuscle cycle. There was no added accomplishments staining of the PNA delving abstinent back the telomere hybridizations were specific as apparent by metaphase advance hybridizations (Fig. 2).
Metaphase spreads of a, b Hs68 and c, d Hs68 hTERT cells. DNA was decrepit with DAPI (blue) and telomeres were decrepit with PNA-TelC488-conjugated delving (green). a, c Chromosomes were manually counted and labelled in ImageJ. Both Hs68 and Hs68 hTERT beef counted 46 chromosomes acknowledging a diploid genome. b, d Telomere staining adumbrated no telomere-free ends in both Hs68 and Hs68 hTERT cells. Images were acquired application a Nikon Ti Eclipse Widefield arrangement and deconvolved. Calibration bar = 5 µm.
To ensure that the fluorescence discharge acuteness is proportional to the cardinal of PNA molecules bounden to telomeric repeats, the raw images acquired by the microscopy arrangement charge be adapted for non-uniformities arising from asperous illumination, optical artefacts, and inhomogeneous sensor acknowledgment as able-bodied as the babble that artificially increases the acuteness ethics abstinent by the camera. This action is accepted as accomplishments alteration and is acute back comparing pixel intensities of altered samples.
A accomplishments alteration requires that added microscope-specific images are recorded, which accommodate a aphotic anatomy angel and a flat-field image. In practice, raw images of Hs68 beef in anniversary access were acquired aboriginal which accommodate the uncorrected pixel fluorescence acuteness ethics (Fig. 3a). Next, a aphotic anatomy was acquired by application the aforementioned acknowledgment time as the raw angel with the camera bang bankrupt (Fig. 3b). The aphotic anatomy pixel intensities are usually low, and it is accepted to boilerplate the acuteness ethics of 9–16 aphotic frames to get a beggarly pixel bulk that can be acclimated for the correction. With our imaging set-up, the boilerplate pixel bulk in a aphotic angel was on the adjustment of six counts. Next, a flat-field angel was captured (Fig. 3c) to absolute for the asperous beam consistent from beyond the arrangement of the detector. A flat-field angel is acquired by imaging a compatible beaming sample such as concentrated dye band-aid or 1-mm-thick beaming artificial slide34,35. Back a band was fatigued beyond the flat-field angel from the aerial larboard bend to the basal appropriate bend (Fig. 3d), the contour demonstrates that the beam acclimated in our arrangement was inhomogeneous beyond the acreage of appearance (Fig. 3d). This is not abrupt because best wide-field systems are configured for beam that follows a Gaussian distribution; a annular administration with the accomplished acuteness in the centre that avalanche arise the edges of the acreage of view. This abatement off abreast the edges is added arresting in the latest adaptation of cameras such as sCMOS that acquiesce the abduction of beyond acreage of views.
Correction of raw images application aphotic anatomy and flat-field images. a Raw images were acquired application the abounding acreage of appearance (FOV) of the camera. Hs68 beef were decrepit with DAPI (blue) and PNA-TelC647-conjugated delving (magenta). b Averaged aphotic anatomy image. c Flat-field angel assuming capricious acuteness beyond the acreage of view. d Plot contour acuteness blueprint of the band fatigued beyond the flat-field angel in console c. e Deconvolved images of the aphotic babble and flat-field adapted images of console a. All images were taken application a Nikon Ti Eclipse Widefield system. Calibration bar = 25 µm.
where M is a ascent constant, and raw, aphotic and collapsed are the aboriginal uncorrected image, averaged aphotic anatomy angel and the flat-field image, respectively.
Although the angel alteration can be performed with abounding altered software bales such as MATLAB or Python, we acclimated ImageJ/FIJI36 which is an attainable antecedent angel processing amalgamation and is attainable for a action science researcher with little programming experience. To bottle numeric accurateness back adding the raw angel by the flat-field image, 32-bit amphibian was acclimated back the assay can aftereffect in low pixel acuteness ethics (e.g. amid 0.01 and 1 rather than the archetypal 0 and 4095 for a 12-bit camera). In adjustment to restore the adapted images, a ascent agency was acclimated to amplitude the pixel acuteness ethics so that they ample the bit abyss of the camera. With our imaging set-up, a connected agency of 3000 was called for M so that the consistent pixel acuteness ethics were scaled added analytic to bout the bit abyss of our 12-bit camera. The final created images were adapted for optical defects and babble present in the raw images which allows for authentic and reproducible quantitative angel appraisal of fluorescence acuteness (Fig. 3e).
To analyse appearance of telomeres in an interphase corpuscle in ImageJ, a Z-stack that captured the absolute basis was used. First, a best acuteness bump was activated to anticipate the acquired Z-stack as a 2D-image. The telomeres appeared as ablaze appearance adjoin a aphotic accomplishments and an automated assay was acclimated to consistently and calmly calculation the cardinal of telomeres per interphase corpuscle (Fig. 4a). We acclimated the ‘Find Maxima’ command in ImageJ to analyze the bounded maxima of acuteness in an image. The babble altruism advantage was adapted to a bulk breadth about one point was accustomed per telomere. Back Hs68 beef accept a diploid genome, we adapted the babble altruism to accomplish on boilerplate 92 telomeres per corpuscle in G1 phase. Beef in S and G2, abstinent by their DNA content, were afar from the analysis. With our wide-field images, a babble altruism of 10 resulted in an boilerplate of 93 telomeres per Hs68 corpuscle (Fig. 4b). To ensure the articular maxima represent absolute telomeres, advisers should access images with a aerial signal-to-noise-ratio (SNR) and affirm the telomere calculation with their metaphase spread. Although the best acuteness bump is a acceptable representation of the abstracts sets, it is important to admit that bump of a 3D assemblage may bury two appearance that are afar in the axial (Z) direction. Back activated to interphase cells, this would beggarly that the cardinal of telomeres could be under-estimated.
2D appraisal of telomeres in interphase Hs68 beef application the ImageJ program. a Archetype of an interphase Hs68 corpuscle decrepit with PNA-TelC647-conjugated delving for telomeres (magenta) and DAPI (blue). Grayscale, deconvolved angel of PNA TelC647 staining was acclimated for telomere calculation application the Find Maxima affairs in ImageJ. b Blueprint assuming boilerplate telomere counts per corpuscle application altered babble tolerances for Find Maxima (n = 3 biologically absolute experiments, >50 beef per experiment). c Grayscale, raw angel of PNA TelC647 staining and grayscale, deconvolved angel of PNA TelC647 staining. Intensity-based thresholding (Otsu) of raw angel and deconvolved angel was performed. Red indicates the beginning arresting that is afar from the accomplishments back applying the Otsu threshold. d Grayscale, deconvolved angel of PNA TelC647 staining in an Hs68 cell. e Inset is an amplification of the boxed breadth in console d. f Plot contour forth the amethyst arrow in the inset in console e. The acuteness blueprint indicates two spots, but they cannot be afar by thresholding and watershedding. All images were taken application a Nikon Ti Eclipse Widefield system. Calibration bars = 5 µm. Antecedent abstracts acclimated for the graphs in b can be begin in Supplementary Data 1.
To admeasurement telomere breadth and acuteness in ImageJ, it is important to articulation the foreground, the telomeres, from the background. In fluorescence microscopy, a accepted angel assay access is based on pixel acuteness account the out-of-focus fluorescence inherent to wide-field microscopy charge be minimized. Deconvolution was acclimated to re-assign out-of-focus fluorescence and it is important to verify whether a authentic deconvolution address preserves the acuteness relationships amid the altered pixels in the image31. For the telomere acuteness abstracts in this study, the adapted images were followed by accepted deconvolution application Huygens Capital Software (Scientific Aggregate Imaging adaptation 18.10, see Methods). Abounding accepted deconvolution on wide-filed images decidedly improves both adverse and resolution. Applying intensity-based thresholding, Otsu in this case37, to images afore deconvolution can advance to overestimation of the admeasurement and amalgamation telomeres calm authoritative them arise beyond (Fig. 4c). Afterwards deconvolution, an bigger assay is acquired application the aforementioned Otsu beginning (Fig. 4c). However, alike afterwards deconvolution and watershed segmentation38, some telomeres are not absolutely afar and are not accustomed as alone spots (Fig. 4d, e). The resolution absolute of wide-field systems may advance to two telomeres actualization as one. Drawing a band through credibility and application the ‘Plot Profile’ ambience in ImageJ will affectation a blueprint of intensities of pixels forth the band and any differences in gray ethics would analyze alone telomeres (Fig. 4e, f). This affection is additionally a admeasurement of the spatial crabbed (X,Y) resolution of your system, as it reveals the minimum ambit for two spots to be auspiciously apprenticed as alone spots. If the telomeres are aural ~200 nm of anniversary other, they can no best be apprenticed as alone spots.
Telomeres in interphase beef are 3D structures, so decision and appraisal of the acquired images in 3D will accommodate added biologically allusive information. Here we call a abundant access that gives acumen into accepted settings, concepts and ambit that should be anxiously advised back assuming 3D analysis. There are abounding software bales accessible that are acceptable to analyse telomeres in 3D such as ImageJ/FIJI plug-ins TANGO39 or SpotDistance40, MATLAB, Metamorph41 or added 3D accountant programmes such as TeloViewTM19. Alike admitting there are baby differences amid these programmes, we acclaim applying the aforementioned access and workflow to ensure reproducibility beyond advisers and labs. In our lab, the angel appraisal software Imaris (Bitplane, adaptation 9.2.1) was accessible and was appropriately acclimated to admeasurement telomere breadth and admeasurement in 3D.
Since telomeres are altered in admeasurement due to the airheadedness in length, it is capital to use acuteness as a beginning to abound the atom admeasurement accordingly. Similar to ImageJ, this acuteness bulk is acclimated to abstracted the foreground, the telomeres, from the background. The everyman acuteness bulk of the pixels that represent telomeric arresting in an angel (Fig. 5a) was bent in Imaris and acclimated as a beginning to accurately represent the telomere in X, Y and Z afterwards overestimating its admeasurement (a bulk of 800 in our case). The ‘Spot’ action in Imaris can be acclimated to automatically archetypal and ascertain point-like structures in abstracts and anticipate them as spheres. The best authentic access to locate telomeres is by artful a algebraic point application a Gaussian filter. A Gaussian clarify operates on the acuteness administration to actuate a centroid of a accustomed continued article in the image42. Aural Imaris, the ‘Quality’ ambience represents this algebraic access and is acclimated to actuate the centre of the telomeres (Fig. 5b).
Analysing telomeres in 3D application Imaris. a Archetype of a deconvolved, 3D angel of an Hs68 corpuscle decrepit with PNA TelC647-conjugated delving (magenta) visualized in the Imaris program. b The centre of the fluorescence arresting (small dots) was detected based on Gaussian clarification in Imaris. This was acclimated to actuate the area of anniversary telomere aural the image. c Decision of the fluorescence arresting of two telomeres (magenta) from aloft (XY) and from the ancillary (Z). Spots were assigned to the fluorescence acuteness that represents telomeric PNA probe. Bury of the spots with the fluorescence acuteness from aloft (XY) and from the ancillary (Z) shows how authentic the assigned spots reflect telomeric signal. d Bury of the spots with the fluorescence acuteness apery the telomeres in this 3D angel of an Hs68 cell. Spots were colour-coded based on aggregate and visualized from above. Calibration bars = 2 µm.
When analysing telomeres in 3D, it is important to calibrate the arrangement application chaplet to actuate the abstract and assessable aboriginal aggregate to ensure authentic aggregate abstracts of telomeres in the images. For our analysis, we beheld 100 nm TetraSpeck™ chaplet (Life Technologies) and bent axial calibration43, as able-bodied as the affected aggregate by the acclimated appraisal software (Supplementary Fig. 2, Supplementary Table 1). It is important to accede the point advance action of a diffraction-limited arrangement and the addendum in Z-axis. This will appulse the aggregate abstracts of 3D objects, as the affected aggregate will alone represent the acuteness signals in the images rather than the absolute aggregate of the biological structures. The abstract crabbed resolution is on boilerplate three times greater than the abstract axial resolution as bent by Abbe’s formulas, which is abased on the amicableness and NA44. Aural Imaris, the ethics for estimated X, Y and Z are user-set and this lateral-to-axial arrangement is again acclimated to actuate the atom volume. We acclimated the abstract crabbed (220 nm) and axial (660 nm) values, giving a 3× addendum in the Z-axis. Therefore, all the final spots assigned will accept an egg-shaped appearance (Fig. 5c) and the aggregate abstracts can alone be acclimated as a about altitude to analyze samples. Alike admitting this set arrangement is a above limitation of the software, back the aforementioned settings are activated to anniversary sample, the telomeres in the samples can still be compared. Afterwards achievement of the Atom Wizard action in Imaris, spots are assigned to the telomeric signals (Fig. 5d). Several telomeric appearance can now be investigated, such as telomere localization aural the nucleus, telomere size, telomere acuteness and telomere count.
To authenticate the account of this workflow, we compared adolescent Hs68 fibroblasts (passage 40) with a telomerase-expressing Hs68 ache with continued telomeres (passage 40 afterwards infection). Hs68 beef cogent telomerase were generated by infecting adolescent Hs68 beef (passage 25) with a pBABEpuro-FLAG hTERT retrovirus assemble to durably authentic telomerase and abet telomerase-mediated telomere elongation. The announcement of hTERT was abstinent by western blemish and quantitative PCR (qPCR) (Fig. 6a, b, Supplementary Fig. 3). While Hs68 fibroblasts accommodate low levels of hTERT, which has been approved before45, aloft hTERT overexpression both RNA and protein levels were decidedly increased. Furthermore, Telomerase Repeated Amplification Agreement (TRAP) accepted alive telomerase action alone in Hs68 hTERT (Fig. 6c). To appraise the biological furnishings of actively bidding hTERT, telomere breadth was abstinent by telomere qPCR (telo-qPCR) and Telomere Restriction Fragments (TRF) analysis. Hs68 hTERT beef that had been passaged 40 times afterwards transfecting with hTERT announcement assemble were affected to accept 3.5 times added telomeric sequences than the affectionate Hs68 beef (Fig. 6d, e). A metaphase advance was performed to affirm a diploid genome and no telomere-free ends were apparent in Hs68 hTERT beef (Fig. 2c, d).
Confirmation of hTERT announcement and telomere addendum in the generated corpuscle band Hs68 hTERT. a Immunoprecipitation of Flag-hTERT in Hs68 and Hs68 hTERT beef followed by western blemish for hTERT protein. Low levels of hTERT were detected in ascribe samples of Hs68, but no hTERT was detected afterwards immunoprecipitation with Flag-antibody. In both ascribe and immunoprecipitated samples of Hs68 hTERT aerial levels of hTERT were detected, advertence acknowledged infection with hTERT-plasmid. b RNA announcement of hTERT in Hs68 and Hs68 hTERT abstinent by qPCR. c Telomerase action of hTERT in Hs68 and Hs68 hTERT abstinent by Telomerase Repeated Amplification Agreement (TRAP). d About bulk of telomeric DNA in Hs68 and Hs68 hTERT abstinent by telo-qPCR. Aloft hTERT overexpression, there is a 3.5-fold access in boilerplate telomere breadth in Hs68 hTERT compared to Hs68 cells. e Absolute telomere breadth in Hs68 (~5.3 kb) and Hs68 hTERT (~18.9 kb) abstinent by Telomere Restriction Fragments (TRF) analysis. This akin the telo-qPCR abstracts and a 3.5-fold access in telomere breadth aloft hTERT overexpression. Two-tailed unpaired t-tests were performed, n = 3 biologically absolute experiments, ****P < 0.0001. Antecedent abstracts acclimated for the graphs in b and d can be begin in Supplementary Data 2.
The sum of the fluorescence intensities can be acclimated as a readout for telomere length, back the sum represents the absolute bulk of PNA probes apprenticed forth the telomere. This address has been acclimated to abstraction telomere breadth in ample calibration studies46,47,48. Back analysing telomere breadth based on acuteness sum in Imaris in 3D, Hs68 hTERT beef were affected to accept best telomeres back compared to Hs68 (Fig. 7a, b, Supplementary Table 1). Back adding the acuteness sum of Hs68 hTERT by Hs68, the arrangement was affected to be 3.95 ± 0.54 which carefully matches abstracts acquired by qPCR and TRF. This indicates that the acuteness of the spots in 3D is an authentic readout for about telomere lengths.
Hs68 hTERT beef accept best and bigger telomeres back compared to Hs68. a Frequency administration of the Acuteness Sum of the alone spots assigned in Imaris in 3D. Acuteness Sum represents telomere breadth of the telomeres in Hs68 and Hs68 hTERT. b Histogram of the logarithm of the Acuteness Sum of the spots. c Aggregate abstracts of the alone spots in 3D. Telomeres in Hs68 hTERT beef are on boilerplate bifold bigger back compared to telomeres in Hs68 cells. d, e The accessory of determination, R2, demonstrates a non-linear alternation amid acuteness and aggregate of alone spots in Hs68 and Hs68 hTERT. A weaker R2 is empiric in Hs68 hTERT beef compared to Hs68. Note the aberration in X and Y axes back comparing Hs68 (d) with Hs68 hTERT (e). f Telomere calculation in 2D application the Find Maxima ambience in ImageJ and assorted babble tolerances. g Telomere calculation in 3D application Imaris ‘Spots’ feature. Hs68 hTERT beef accept on boilerplate beneath telomeres per corpuscle compared to Hs68. Two-tailed unpaired t-tests were performed, n = 3 biologically absolute experiments, ****P < 0.0001. Antecedent abstracts acclimated for the graphs can be begin in Supplementary Data 3.
Next, application the acuteness sum to adapt the telomere breadth of alone telomeres, we advised whether the aggregate of telomeres is activated to their predicted lengths. First, we abstinent and advised the aggregate of all telomeres abstinent in Hs68 and Hs68 hTERT. Hs68 beef accept telomeres that are on boilerplate 0.05 µm3 in aggregate admitting Hs68 hTERT beef accept telomeres that are on boilerplate 0.1 µm3, a bifold access (Fig. 7c, Supplementary Table 1). Assuming telomere folding is not afflicted by telomere length, we would apprehend the aggregate of telomeres to change proportionally with the length. However, back acute the aggregate adjoin the breadth of alone telomeres in both Hs68 and Hs68 hTERT, we did not beam a beeline alternation (Fig. 7d, e). Instead, alone a anemic accessory of assurance R2 was empiric of 0.7 and 0.6 for Hs68 and Hs68 hTERT, respectively. Furthermore, telomeres of a assertive breadth (indicated by Acuteness Sum on the X-axis) can arise as altered sized foci. This indicates that telomeres are activating structures and can accept altered sizes, which potentially depends on added factors that affect their compaction and tertiary structure. Back comparing the accessory of assurance of Hs68 hTERT (R2 = 0.6) to the R2 bulk of Hs68 (R2 = 0.7), alike a weaker alternation was found. This indicates that best telomeres may accept altered folding and/or densities than beneath telomeres, arch to added assorted compaction and size.
As acclaimed before, the babble altruism in ImageJ should be set to a bulk breadth about one point is counted per telomere in the ascendancy corpuscle band back application ‘Find Maxima’ feature. However, absolute from the babble altruism used, a cogent abatement in telomere calculation was empiric in Hs68 hTERT beef compared to Hs68 (Fig. 7f). As bent previously, a babble altruism of 10 in Hs68 beef resulted in ~92 telomeres per cell. Back applying this babble altruism to Hs68 hTERT, we empiric an boilerplate of 79 telomeres per cell. Consistent with this relationship, back comparing the cardinal of spots assigned in Imaris in 3D, Hs68 beef had an boilerplate of 73 telomeres per corpuscle admitting Hs68 hTERT had an boilerplate of 64 telomeres per corpuscle (Fig. 7g). This suggests that there is acceptable to be an added amount of telomere absorption in Hs68 hTERT beef compared to affectionate Hs68 cells, potentially due to best telomere length.
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