To evaluate the capability of MesoTIRF for dual-wavelength imaging including an assessment of the uniformity of illumination, a fixed HeLa cell specimen was prepared, using an anti-paxillin antibody conjugated to the fluorescent secondary antibody Alexa Fluor Plus 594 and fluorescein phalloidin that stains F-actin. For each sequential image, an exposure time of 2 s and a camera gain of 30× was used.
To evaluate the improvement in SBR in MesoTIRF compared with WF epi, the same specimen was imaged but only the anti-paxillin conjugated to Alexa Fluor Plus 594 was excited. To measure the SBR, the machine learning algorithm collection Trainable Weka2222. I. Arganda-Carreras, V. Kaynig, C. Rueden, K. W. Eliceiri, J. Schindelin, A. Cardona, and H. S. Seung, “ Trainable Weka segmentation: A machine learning tool for microscopy pixel classification,” Bioinformatics 33, 2424–2426 (2017). https://doi.org/10.1093/bioinformatics/btx180 in ImageJ was used to identify and segment fluorescent objects from both WF epi and MesoTIRF images. A selection mask was extracted from this segmentation, which enabled us to derive the mean detected signal in a cell from the raw images using ImageJ.2020. C. T. Rueden, J. Schindelin, M. C. Hiner, B. E. DeZonia, A. E. Walter, E. T. Arena, and K. W. Eliceiri, “ ImageJ2: ImageJ for the next generation of scientific image data,” BMC Bioinf. 18, 529 (2017). https://doi.org/10.1186/s12859-017-1934-z The background signal was calculated for each image by selecting three background regions of interest, calculating the mean intensity, and averaging these measurements. This workflow was carried out for six ROIs separated by at least 0.5 mm across the full FOV to evaluate variations in SBR, and the uniformity of illumination.To demonstrate MesoTIRF in another cell type, the human mesothelial cell line MeT-5A was fixed and labeled with antibodies against paxillin and tubulin, which were conjugated to Alexa Fluor 488 and Alexa Fluor Plus 594, respectively. These (adhesion and cytoskeletal) proteins are within the reach of an evanescent field. These dataset is included in the supplementary material.Cell culture, fluorescent labeling, and specimen preparation methods are included in the supplementary material alongside quantification of focal adhesions imaged under WF epi and MesoTIRF.Figure 2 shows a comparison of WF epi and MesoTIRF images of dual-labeled fixed 3T3-L1 cells prepared with fluorescent staining for nuclei (green) and paxillin (magenta). The full FOV WF epi image is shown in Fig. 2(a), with a region of interest (ROI) indicated by a yellow box that is digitally zoomed in 2B. Figure 2(c) shows the same area of the specimen imaged using dual-wavelength MesoTIRF, with the same ROI expanded in 2D.Using WF epi, the cell nuclei are clearly visible in Fig. 2(b). These nuclei disappear, as expected, when imaged with MesoTIRF as shown in 2D, thus confirming that the evanescent wave in MesoTIRF is restricted to a shallow depth close to the coverslip and does not penetrate sufficiently deep into the sample to excite fluorescence from the labeled nuclei. Nonspecific binding or binding of the anti-paxillin antibody to cytosolic protein is apparent when imaged with WF epi, but this fluorescence signal also disappears when using MesoTIRF illumination. This is further evidenced by the intensity profiles through two neighboring nuclei [Fig. 2(e)] and through three neighboring focal adhesions [Fig. 2(f)] for both the WF epi image (teal) and MesoTIRF (dark red). The difference in background is clearly evident, with MesoTIRF yielding a 4.2-fold reduction in background through the neighboring focal adhesions over WF epi, with a less noisy baseline than that of the WF epi image. While the focal adhesions are still visible in WF epi, the contrast enhancement afforded by MesoTIRF allows for the elongated features to be easily distinguished from background. An SBR improvement of 4.84×/3.9×/3.87× was observed in focal adhesions 1, 2, and 3 [measured from peaks in Fig. 2(f) left to right], respectively, when switching from imaging with WF epi to MesoTIRF.Furthermore, there is negligible nuclear signal in the MesoTIRF image [Fig. 2(e)] because, as discussed previously, the 86° incident beam (resulting in a calculated evanescent field depth of 56 nm) does not penetrate the cell specimen deep enough to excite the nuclear stain. Using the Imaris based “Surfaces” feature in the nuclear channel, 743 cells were counted in this single image. For the purposes of presentation, each image presented in Fig. 2 underwent the “Contrast Limited Adaptive Histogram Equalization (CLAHE)” local contrast adjustment function in ImageJ.2020. C. T. Rueden, J. Schindelin, M. C. Hiner, B. E. DeZonia, A. E. Walter, E. T. Arena, and K. W. Eliceiri, “ ImageJ2: ImageJ for the next generation of scientific image data,” BMC Bioinf. 18, 529 (2017). https://doi.org/10.1186/s12859-017-1934-z However, all analysis has been performed on raw image data.The application of MesoTIRF for imaging of dual-labeled specimens is shown in Fig. 3. Figure 3 shows a 4.4 × 3.0 mm2 FOV dual-color MesoTIRF image, following application of CLAHE, with focal adhesions in magenta and F-actin in cyan. Yellow boxes show digitally zoomed images of six separate ROIs separated by a minimum distance of 0.5 mm. In all images, focal adhesions and the F-actin network adjacent to the basal cell membranes are clearly visible. We note that there is a less than a 50% decrease in fluorescence signal from the center to the edge of the imaged field, which we attribute to the Gaussian intensity profile of the illumination yielded from the optics chosen in Fig. 1.Image quality analysis, quantification of resolvable detail with each illumination technique, and further MesoTIRF cell imaging is included in the supplementary material. Five line intensity profiles through neighboring fluorescent features imaged with WF epi and MesoTIRF served to measure the contrast improvement of MesoTIRF alongside providing topographical analysis of the focal adhesion features (Fig. S1). Uniformity of the MesoTIRF illuminator, using image data in Fig. 3, was quantified by counting the number of focal adhesions across six ROIs across the full image FOV (Fig. S2). Following on from this biological measure of uniformity, illumination uniformity was further examined using fluorescent test slides and measuring the cross section of each illumination technique with each utilized excitation wavelength (Fig. S3). Figure S4 shows a further example of two-color MesoTIRF imaging with the cell type MeT-5A labeled for paxillin and tubulin.We have demonstrated MesoTIRF imaging of a range of different biological samples. Coupling a custom prism TIRF illuminator with the Mesolens1212. G. McConnell, J. Trägårdh, R. Amor, J. Dempster, E. Reid, and W. B. Amos, “ A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout,” eLife 5, e18659 (2016). https://doi.org/10.7554/eLife.18659 provides an unprecedented combination of a large FOV with high spatial resolution and high contrast image quality. The optical throughput of the Mesolens is 25 times greater than a commercial objective lens with a low magnification.1212. G. McConnell, J. Trägårdh, R. Amor, J. Dempster, E. Reid, and W. B. Amos, “ A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout,” eLife 5, e18659 (2016). https://doi.org/10.7554/eLife.18659 This presents an advantage for MesoTIRF imaging, which enables lower optical power specimen illumination with corresponding reductions in photobleaching and phototoxicity. MesoTIRF utilized the comparatively cheap prism illumination method, allowing for ease when changing evanescent field depth by varying the incidence angle of the incident beam.In our confirmation of TIRF illumination using the specimen that was dual-labeled with a nuclear stain and an antibody against the focal adhesion protein paxillin residing in the plasma membrane, we note that the position of the nuclei is dependent on where in their life-cycle the 3T3-L1 cells were at the point of formaldehyde fixation.2323. M. Webster, K. L. Witkin, and O. Cohen-Fix, “ Sizing up the nucleus: Nuclear shape, size and nuclear-envelope assembly,” J. Cell Sci. 122, 1477–1486 (2009). https://doi.org/10.1242/jcs.037333 However, we expect that the nuclear envelopes of each imaged cell was distal from the basal cell membrane,2323. M. Webster, K. L. Witkin, and O. Cohen-Fix, “ Sizing up the nucleus: Nuclear shape, size and nuclear-envelope assembly,” J. Cell Sci. 122, 1477–1486 (2009). https://doi.org/10.1242/jcs.037333 and therefore, outside the reach of an evanescent field from the MesoTIRF illuminator.The ability of the MesoTIRF modality to capture fine details and the contrast improvement over WF epi was examined using a fixed mammalian cell line labeled for the focal adhesion component paxillin.1919. C. E. Turner, J. R. Glenney, and K. Burridge, “ Paxillin: A new vinculin-binding protein present in focal adhesions,” J. Cell Biol. 111, 1059–1068 (1990). https://doi.org/10.1083/jcb.111.3.1059 Using the intensity signals through neighboring focal adhesions, an average 4.2-fold improvement in SBR was measured in MesoTIRF over WF epi, with the improvement in contrast allowing for more focal adhesions to be resolved with this novel modality (Fig. 2), while additionally yielding some topographical information (Fig. S1).The dropoff in the intensity from the center to the edge of the MesoTIRF FOV was to be expected for an evanescent field generated using a Gaussian beam. This can be corrected either optically with beam shaping components to change the Gaussian profile to, for example, a top-hat,2424. A. Möhl, S. Wickenhagen, and U. Fuchs, “ Gauss to top-hat beam shaping with aspheres,” Proc. SPIE 9741, 974102 (2016). https://doi.org/10.1117/12.2209522 or computationally, with algorithms, such as flat field correction,2525. M. Model, “ Intensity calibration and flat-field correction for fluorescence microscopes,” Curr. Protoc. Cytom. 68, 10.14.1 (2014). https://doi.org/10.1002/0471142956.cy1014s68 a commonplace post-processing technique for many imaging modalities. However, as evident from the chosen ROIs in Fig. 3, the structural detail resolvable even in these dimmer peripheral areas remains of the quality expected of TIRF, with the same low background and individual punctate focal adhesions and spindled actin filaments that would be blurred by fluorescent signal excited from deeper in the cell in WF epi. A quantitative analysis of this uniformity, both from the biological specimen image provided in Fig. 3 and with a uniform non-biological test specimen, is included in the supplementary material (Figs. S2 and S3).Excitation wavelengths for MesoTIRF are presently limited to the two discussed here by the large diameter Pinkel-type custom filters1515. D. Pinkel, J. Gray, R. Segraves, F. Waldman, B. Trask, L. C. Yu, D. Eastmond, and P. Dean, “ Fluorescent nucleic acid hybridization methods,” Proc. SPIE 1063, 123 (1989). https://doi.org/10.1117/12.951898 used for fluorescence detection. Additional custom filters would allow this to be extended for further wavelengths.Mesolens data are rich in information,2626. M. Shaw, R. Claveau, P. Manescu, M. Elmi, B. J. Brown, R. Scrimgeour, L. S. Kölln, G. McConnell, and D. Fernandez-Reyes, “ Optical mesoscopy, machine learning, and computational microscopy enable high information content diagnostic imaging of blood films,” J. Pathol. 255, 62–71 (2021). https://doi.org/10.1002/path.5738 but we recognize that an imaging rate of 0.2 Hz for MesoTIRF is insufficient for several applications in vitro such as cell signaling studies as reported by Crites et al.2727. T. J. Crites, L. Chen, and R. Varma, “ A TIRF microscopy technique for real-time, simultaneous imaging of the TCR and its associated signaling proteins,” J. Visual. Exp. 61, 3892 (2012). https://doi.org/10.3791/3892 However, with recent innovations in camera technologies, notably the development of cameras using large, high resolution 250 Mpixel sensors, such as the Canon 2U250MRXSAA CMOS sensor, tenfold higher imaging speeds (2.4 fps) can be achieved by avoiding the need for chip shifting. Additionally, sensor shifting technology has advanced in recent years, and a new sensor shifting camera providing a 604 MPixel image with a 1.5 fps imaging speed is now commercially available (VNP-604MX-MC-6-H, Vieworks). In combination with environmental control, this will offer opportunities to study faster dynamic processes, for example, the action of fast-acting antimicrobial peptides2828. A. K. Buck, D. E. Elmore, and L. E. Darling, “ Using fluorescence microscopy to shed light on the mechanisms of antimicrobial peptides,” Future Med. Chem. 11, 2447–2460 (2019). https://doi.org/10.4155/fmc-2019-0095 or imaging of calcium transients in the plasma membrane.2929. P. Toglia, G. Ullah, and J. E. Pearson, “ Analyzing optical imaging of Ca2+ signals via TIRF microscopy: The limits on resolution due to chemical rates and depth of the channels,” Cell Calcium 67, 65–73 (2017). https://doi.org/10.1016/j.ceca.2017.08.010MesoTIRF may have applications in high-content screening1010. D. A. Coucheron, Ø. Ivar Helle, C. I. Øie, J. C. Tinguely, and B. S. Ahluwalia, “ High-throughput total internal reflection fluorescence and direct stochastic optical reconstruction microscopy using a photonic chip,” J. Visual. Exp. 2019, e60378. https://doi.org/10.3791/60378 or wound healing models,3030. S. Sen-Britain, D. M. Britain, W. L. Hicks, and J. A. Gardella, “ TOF-SIMS and TIRF microscopy investigation on the effects of HEMA copolymer surface chemistry on spatial localization, surface intensity, and release of fluorescently labeled keratinocyte growth factor,” Biointerphases 14, 051003 (2019). https://doi.org/10.1116/1.5119871 where large cell populations must be imaged to obtain statistically significant results. However, at present, MesoTIRF is only compatible with imaging at room temperature, as there is no environmental imaging chamber that is compatible with the Mesolens. We are presently considering chamber designs that would be suitable for long-term imaging applications, including MesoTIRF.A present limitation of MesoTIRF is the numerical aperture of the Mesolens; at 0.47, this is much lower than a typical TIRF lens, and, hence, the lateral resolution is around threefold poorer than that of a commercial objective TIRF microscope. With the principle of MesoTIRF now proven, one solution would be to increase spatial resolution using structured illumination,31,3231. P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. Gustafsson, “ Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6, 339–342 (2009). https://doi.org/10.1038/nmeth.132432. J. Roth, J. Mehl, and A. Rohrbach, “ Fast TIRF-SIM imaging of dynamic, low-fluorescent biological samples,” Biomed. Opt. Express 11, 4008 (2020). https://doi.org/10.1364/BOE.391561 introducing the possibility of super-resolution MesoTIRF-SIM. However, achieving SIM on the Mesolens is not trivial and would require either further optics in the MesoTIRF path to impose variable modulation patterns on the incident excitation beam or utilizing computational methods such as blind-SIM3333. R. Ayuk, H. Giovannini, A. Jost, E. Mudry, J. Girard, T. Mangeat, N. Sandeau, R. Heintzmann, K. Wicker, K. Belkebir, and A. Sentenac, “ Structured illumination fluorescence microscopy with distorted excitations using a filtered blind-SIM algorithm,” Opt. Lett. 38(22), 4723–4726 (2013). https://doi.org/10.1364/OL.38.004723 algorithms. This would facilitate applications in single molecule localization microscopy in cell specimens approximately two orders of magnitude larger than current technology can image. At present, we are again limited by chip-shifting camera technology, but we are carefully following developments in this field.See the supplementary material for cell specimen preparation, quantification of fine spatial detail visible under MesoTIRF illumination, quantification of the uniformity of the illumination, and additional cell membrane protein MesoTIRF imaging.S.F. was supported by an iCASE award from the Engineering and Physical Sciences Research Council and the National Measurement System of the UK’s Department for Business, Energy, and Industrial Strategy. L.K. was supported by the Engineering and Physical Sciences Research Council and the Medical Research Council (MRC) for Doctoral Training in Optical Medical Imaging (No. EP/L016559/1). The work ongoing in the Gram Hansen lab is supported by a University of Edinburgh Chancellor’s Fellowship, Worldwide Cancer Research (No. 19-0238) and the June Hancock Mesothelioma Research Fund. M.S. is funded by the National Measurement System of the Department for Business, Energy, and Industrial Strategy. G.M. was supported by the Biotechnology and Biological Sciences Research Council (Nos. BB/P02565X/1 and BB/T011602/1) and the MRC (No. MR/K015583/1). Both G.M. and W.B.A. are partially supported by the Leverhulme Trust.
Conflict of Interest
The authors have no conflicts to disclose.
Author Contributions
Shannan Foylan: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Funding acquisition (supporting); Investigation (lead); Methodology (equal); Validation (equal); Visualization (equal); Writing – original draft (lead); Writing – review & editing (equal). William Bradshaw Amos: Methodology (equal); Visualization (supporting); Writing – original draft (supporting); Writing – review & editing (equal). John Dempster: Data curation (equal); Methodology (equal); Resources (equal); Software (lead); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). Lisa Koelln: Conceptualization (equal); Methodology (equal); Writing – review & editing (equal). Carsten Gram Hansen: Conceptualization (supporting); Funding acquisition (equal); Project administration (equal); Resources (equal); Writing – review & editing (equal). Michael James Shaw: Conceptualization (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Supervision (equal); Writing – original draft (equal); Writing – review & editing (equal). Gail McConnell: Conceptualization (lead); Funding acquisition (lead); Methodology (lead); Project administration (lead); Resources (lead); Supervision (lead); Validation (equal); Writing – original draft (equal); Writing – review & editing (equal).
The data that support the findings of this study are available within the article and its supplementary material.REFERENCES
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