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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 3 — Jan. 30, 2012
  • pp: 3311–3324
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Highly confined surface imaging by solid immersion total internal reflection fluorescence microscopy

Lin Wang, Cvetelin Vasilev, Daniel P. Canniffe, Luke R. Wilson, C. Neil Hunter, and Ashley J. Cadby  »View Author Affiliations


Optics Express, Vol. 20, Issue 3, pp. 3311-3324 (2012)
http://dx.doi.org/10.1364/OE.20.003311


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Abstract

We report the use of a high-refractive-index aplanatic solid immersion lens (ASIL) in total internal reflection fluorescence (TIRF) microscopy. This new solid immersion total internal reflection fluorescence (SITIRF) microscopy allows highly confined surface imaging with a significantly reduced imaging depth compared with conventional TIRF microscopy. We explore the application of a high refractive index, low optical dispersion material zirconium dioxide in the SITIRF microscope and also introduce a novel system design which enables the SITIRF microscope to work either in the epi-fluorescence or TIRF modes with variable illumination angles. We use both synthetic and biological samples to demonstrate that the imaging depth in the SITIRF microscope can be confined to a few tens of nanometers. SITIRF microscopy has the advantages of performing highly selective imaging and high-resolution high-contrast imaging. Potential applications in biological imaging and future developments of SITIRF microscopy are proposed.

© 2012 OSA

1. Introduction

Solid immersion lenses (SILs) can be used in optical microscopy to improve spatial resolution [1

1. S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57(24), 2615–2616 (1990). [CrossRef]

]. In this technique, the gap between the sample and the objective lens is filled by solid material with a high refractive index n, which increases the numerical aperture (NA). For the combination of a traditional hemispherical SIL (HSIL), i.e. a hemispherical lens with a thickness equal to its radius, and a conventional objective lens, the NA can be increased by a factor of n [1

1. S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57(24), 2615–2616 (1990). [CrossRef]

] compared to that of the conventional objective lens alone; while, for the combination of an aplanatic SIL (ASIL), i.e. a truncated ball lens of thickness r + r/n where r is the radius, and a conventional objective lens, it can be increased by a factor of n2 up to a maximum value of n [2

2. B. D. Terris, H. J. Mamin, D. Rugar, W. R. Studenmund, and G. S. Kino, “Near-field optical data storage using a solid immersion lens,” Appl. Phys. Lett. 65(4), 388–390 (1994). [CrossRef]

]. Thus generally speaking the application of ASILs in an optical microscope can achieve higher NA and resolution than that of HSILs. Besides, in contrast to HSILs, ASILs can provide a larger effective field of view (FOV) taking into account the tolerance of geometrical aberrations induced by the SILs [3

3. J. Zhang, C. W. See, and M. G. Somekh, “Imaging performance of widefield solid immersion lens microscopy,” Appl. Opt. 46(20), 4202–4208 (2007). [CrossRef] [PubMed]

]; therefore in our opinion ASILs are more preferable than HSILs in the development of wide-field high-resolution optical microscopy.

Another promising case that may take advantage of high NA values in SIF microscopy is objective-based total internal reflection fluorescence (TIRF) microscopy [9

9. A. L. Stout and D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28(24), 5237–5242 (1989). [CrossRef] [PubMed]

]. TIRF microscopy is a wide-field high-resolution fluorescence microscopy used to image the surface of cells by utilizing a thin field of illumination. This illumination is generated by shining a tilted beam, commonly from a laser source, to the interface between the cells and the substrate, which creates a shallow evanescent field that excites fluorophores on the cell surface. TIRF microscopy is widely used in the study of cell-substrate contacts [10

10. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]

], membrane dynamics [11

11. J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008). [CrossRef] [PubMed]

], single molecule imaging [12

12. T. Yanagida, Y. Sako, and S. Minoghchi, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol. 2(3), 168–172 (2000). [CrossRef] [PubMed]

] and endocytic and exocytotic events [13

13. J. Z. Rappoport and S. M. Simon, “Real-time analysis of clathrin-mediated endocytosis during cell migration,” J. Cell Sci. 116(5), 847–855 (2003). [CrossRef] [PubMed]

]. TIRF is often favored as a way of imaging a very shallow depth from the cell-substrate surface in order to gain high sensitivity or/and high selectivity. In objective-based TIRF microscopy the imaging depth is regulated by the NA of the objective lens. As reported in [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

], SIF microscopy is able to achieve an NA much higher than those of conventional oil immersion TIRF microscopy objective lenses, so it is promising to explore TIRF microscopy based on a SIF microscope platform.

In this paper, we report a new approach combining high NA SIF microscopy and TIRF microscopy, which we refer to as solid immersion total internal reflection fluorescence (SITIRF) microscopy, and the highly confined surface imaging capability in this new microscopic technique.

2. Theory

TIRF microscopy employs total internal reflection (TIR) illumination, as shown in Fig. 1
Fig. 1 TIR illumination scheme. When the incident angle of the incoming wave is larger than the critical angle θc, the wave is totally internally reflected. The evanescent wave propagates in the x direction and decays exponentially in the low-index (n2) material in the z direction.
, to selectively illuminate and excite fluorophores in a thin optical plane close to the interface between the two materials. TIR occurs at an interface formed by two media with different indices of refraction. According to Snell’s law, for light propagating from an optically denser (refractive index: n1) to a less-dense (refractive index: n2) medium, i.e., n1> n2, if the incident angle of the beam is larger than the critical angle θc, which is defined by:
θc=arcsin(n2/n1)
(1)
the beam will be totally internally reflected in the denser medium without refraction in the less-dense one. While the evanescent wave from the incident beam keeps propagating parallel to the interface and then penetrates through the evanescent field, the evanescent wave decays exponentially from the interface into the low-index medium as [14

14. D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng. 13(1), 247–268 (1984). [CrossRef] [PubMed]

]:
I(z)=I(0)exp(z/d)
(2)
where I(0) is the intensity at the interface, z is the perpendicular distance from the interface and d is the penetration depth, defined as the distance over which the intensity of the evanescent field decays to 1/e of its value at the interface. The exponential drop in the evanescent wave makes thin optical slices attainable in TIRF microscopy, and as such the background fluorescence from outside the area of interest can be reduced significantly. When utilized with membrane-specific fluorophores, TIRF microscopy is capable of visualizing the structures in and around the cell membrane with a high signal-to-noise ratio. In conventional fluorescence microscopes, signals from these structures are usually overwhelmed by fluorescent emission from background objects. The penetration depth d is determined by:
d=λ4πn12sin2θn22
(3)
where λ is the wavelength of the incident light, θ is the incident angle. If we refer to the NA used for the illumination as illumination NA (NAill), the above equation can be rewritten as:

d=λ4πNAill2n22
(4)

From this equation we can see that, in order to confine penetration depth to a small amount at the interface, it is essential to have an illumination NA that is significantly greater than the refractive index of the medium surrounding the sample, i.e. NAill >> n2.

In a practical TIRF microscope used in cell imaging, the most common medium around cell samples is water, with an index of 1.33. In theory any objective lens providing a NA higher than this value is capable of generating TIR; however, using objective lenses with NAs just above 1.33 by a small margin will produce TIR at an angle insufficiently larger than the critical angle, and the evanescent field in this case will be deep. Typical TIRF microscopy objective lenses made by the major manufacturers have the NAs in the range of 1.45-1.49, and the highest NA available in market is 1.65 [15

15. P. Török and F. J. Kao, Optical Imaging and Microscopy: Techniques and Advanced Systems,87 of Springer series in optical sciences (Springer, 2007).

] but unfortunately that objective requires the use of expensive n = 1.78 coverslips and special n = 1.78 coupling oil which is volatile and leaves a crystalline residue. Recently it was demonstrated that SIF microscopy employing an ASIL can provide an illumination NA as high as 2 [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

], and ASIL-based fluorescence microscopy is generally adaptable to other types of wide-field high-resolution fluorescence microscopy. If the TIR illumination scheme is used in ASIL-based fluorescence microscopy, highly confined surface imaging is potentially achievable owing to the high NA performance in SIF microscopy.

As reported in [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

] the NA in SIF microscopy employing an ASIL is effectively determined by the index of the ASIL material, and the illumination NA of 2 was demonstrated by applying an ASIL made of high-index optical glass (S-LAH79) that has a refractive index of nd = 2.003 and an Abbe number of νd = 28.27. The major issue in the application of this ASIL is the appearance of severe chromatic aberration (CA) due to the strong dispersion in the optical glass, as the low Abbe number indicated. In practice a very narrow band emission filter, with a 3 nm spectral bandwidth (full width at half maximum, FWHM), was used to reduce CA to a negligible level and maintain high-resolution performance. However the narrow bandwidth of this filter hindered the fluorescence imaging because of the lack of detectable emitted photons. It is desirable to find optical materials with less dispersion, i.e. a higher Abbe number, to fabricate ASILs. In our current research, zirconium dioxide (ZrO2), a type of crystalline material with nd = 2.215 and νd = 51.11, was used to fabricate ASILs. In contrast to S-LAH79 optical glass, ZrO2 features both higher index and lower dispersion, which could offer many benefits, including (a) the elimination of CA, which allows the use of wide band emission filters, with a consequent improvement in the efficiency of fluorescence detection; (b) a higher index, leading to a higher NA and gains in imaging resolution, as the illumination and imaging beams share the same objective lens in an objective-based TIRF microscope; (c) a higher illumination NA, which creates a shallower evanescent field.

In a SITIRF microscope employing an ASIL made of ZrO2 the highest achievable illumination NA equals the index of ZrO2, which is 2.230 at a wavelength of 473 nm. The solid blue curve depicted in Fig. 2
Fig. 2 Simulated intensity decays along the z direction from the ASIL-air (blue curves) and ASIL-water (red curves) interfaces with illumination NA values of 2.2 and 1.45. Theoretical penetration depths are also marked by circles in each case. An illumination wavelength of 473 nm is considered.
shows how the intensity decays along the z direction from the ASIL-air interface, according to Eq. (2). The dashed blue curve in the same figure displays the intensity decay from an illumination NA of 1.45, a characteristic NA value in conventional TIRF microscopes. The decay of the evanescent wave in the SITIRF microscope is more pronounced than in a conventional TIRF microscope, showing that a highly confined excitation depth is theoretically obtainable in SITIRF microscopy. Quantitatively from Eq. (4) we know that the penetration depths are 19 nm and 36 nm in the SITIRF and conventional TIRF microscopes, respectively, which are marked by the circles in Fig. 2. The intensity decays happening at an ASIL-water interface with different illumination NAs are depicted by red curves in Fig. 2, and in this case the penetration depths are 21 nm and 65 nm in the SITIRF and conventional TIRF microscopes, respectively. From these curves we know SITIRF microscopy can perform highly confined evanescent field illumination at similar level with either air medium or water medium, but conventional TIRF microscopy shows an obviously different penetration depth with different media. Therefore we can conclude SITIRF microscopy has a more general use when implementing surface imaging, and for this reason the following experiments were carried out in air.

To investigate the CA performance of ZrO2 ASILs and to guide the choices of emission filters that can be used in practice, we used the optical design software ZEMAX (ZEMAX Development Corporation) to simulate the CA of 3 mm diameter ASILs made of ZrO2 at a central wavelength of 473 nm . The on-axis spot diagrams with an Airy disk reference from the simulations are shown in Fig. 3
Fig. 3 Spot diagrams showing PSF with Airy disk reference at the central wavelength (black circle) of 473 nm for an isotropic point source at the aplanatic point of a 3 mm ZrO2 ASIL. (a) The PSF for ASIL imaging for three wavelengths: 468 (blue crosses), 478 nm (red squares) and 473 nm (green crosses) which is focused at the paraxial focal point and therefore forms a diffraction-limited image corresponding to the Airy disk. (b) The PSF for ASIL imaging for three wavelengths: 463 (blue crosses), 483 nm (red squares) and 473 nm (green dots) which is focused at the paraxial focal point and therefore forms a nearly diffraction-limited image corresponding to the Airy disk.
. The spot diagrams in Fig. 3(a) show that with a 10 nm imaging spectral width, i.e. from 468 nm to 478 nm, the geometric point spread function (PSF) at all wavelengths is well within the Airy disk when the central wavelength is focused at the paraxial focal point, indicating diffraction-limited resolution. If the imaging spectral width is extended to 20 nm, i.e. from 463 nm to 483 nm, the resolution is still close to diffraction-limited performance (Fig. 3(b)). Our simulations also show that the nearly diffraction-limited imaging performance can be maintained within a 30 μm diameter FOV. According to these simulation results, we chose to use emission filters with spectral bandwidths no wider than 20 nm in our experiments in order to achieve high-resolution imaging.

3. Experimental methods

The optical arrangement of SITIRF microscopy is shown in Fig. 4
Fig. 4 Schematic of the SITIRF microscope system. A mirror and a steering lens were mounted on a motorized translation stage to change the incident illumination angle. The dark blue/red shading illustrates the Köhler illumination beam from either of the two lasers, the light blue shading illustrates LED illumination beam, the green shading indicates the fluorescent light forming the specimen images, and the red dashed lines represent the fluorescent light forming the BFP images.
. The fundamental construction of this system is similar to the SIF microscope system described in [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

], in which an epi-fluorescence configuration was applied, while to manipulate the incident illumination angle a mirror and a steering lens, as enclosed by the dashed box in Fig. 4, were mounted on a motorized translation stage (PT1/M-Z8, Thorlabs) to allow radial positioning of the illumination focusing spot on the back focal plane (BFP). When the focusing spot was on-axis, a conventional Köhler illumination was formed; once the focusing spot was off-axis to produce a tilted but collimated illumination above the critical angle, a TIR illumination was generated. This configuration allowed us not only to conveniently transform the system between an epi-fluorescence microscope and a SITIRF microscope, but also to exert fine control over the illumination NA because the radial position of the focused beam on the BFP has a linear relationship with the illumination NA [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

]:
rspot=feffNAill
(5)
where rspot is the radial distance from the geometrical center of the BFP to the focusing spot, and feff is the effective focal length of the ASIL-objective, determined by [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

]:

feff=fn2
(6)

Here, f is the focal length of the conventional objective lens and n is the index of ASIL material, so we can conclude the effective focal length in our case was about 0.8 mm.

In this system, a 0.55 NA long working distance objective lens (50 × Plan Apo Infinity-Corrected Long WD Objective, Mitutoyo, focal length 4 mm) and a 3 mm diameter ASIL made of ZrO2 were used to form an ASIL-objective. The ASIL was firstly purchased from Knight Optical Ltd as custom made ZrO2 ball lenses, and then grinded and polished to the aplanatic configuration. The distance between the convex surface of the ASIL and the front surface of the Mitutoyo objective was 8.2 mm and the medium between them was air. The illumination NA of the ASIL-objective was determined by the index of the ASIL material. In our case, a 473 nm diode pumped solid state (DPSS) laser (MBL-III-473, Laser2000) and a 632.8 nm Helium-Neon (He-Ne) laser (31-2140-000, Coherent) were available for excitation; the indices of ZrO2 at these wavelengths are 2.230 and 2.208, respectively, so we conclude that the maximum available illumination NA was about 2.2. For bright-field imaging, an LED (LXHL-LE5C, Lumileds) with a central wavelength of 505 nm was also used as an additional light source. A multiband dichroic filter (Z470/635rpc, Chroma) was employed to reflect either of the laser beams into the objective, while it was also used as a 50/50 beam splitter for the bright-field LED illumination. A group of emission filters was mounted on a filter wheel for easy selection of imaging wavelength. In order to maintain nearly diffraction-limited imaging and to conform to the simulation results (Fig. 3) all the emission filters had a spectral bandwidth of less than 20 nm. The imaging path was divided by a beam splitter to enable the simultaneous capture of sample images and monitoring of BFP distribution, recorded by a CCD camera (ORCA-ERG, Hamamatsu) and a CMOS camera (DCC1545M, Thorlabs), respectively. The FOVs for sample and BFP imaging were approximately 29 × 22 µm and 10 × 8 mm.

The ASIL was firstly cleaned by immersing it in Acetone and sonicating it for 15 minutes, and then cleaned by immersing it in Piranha solution for 10 minutes. To enhance cell adhesion, the flat surface of the ASIL was coated by Poly-L-Lysine. After cleaning the ASIL was glued on a brass holder and then mounted on a kinematic mount in the system. Once the ASIL was finely aligned the samples could be directly drop cast on its flat surface for imaging.

4. Experiments and results

As mentioned previously, the SITIRF system could be conveniently switched between epi-fluorescence mode and TIRF modes, and the illumination NA in TIRF mode could be finely controlled by the motorized translation stage, so samples could be imaged in three different modes: (a) epi-fluorescence, (b) TIRF with an illumination NA of 1.45 typical of commercial TIRF microscopes, designated conventional NA TIRF mode, and (c) TIRF with an illumination NA of 2.2, designated high NA TIRF mode. The first two modes were used to mimic the performance of an epi-fluorescence microscope and a conventional TIRF microscope, respectively. Comparison of images captured in these three different modes revealed the effective imaging depths in epi-fluorescence, conventional TIRF and SITIRF microscopes. The goal of this work is to demonstrate that the SITIRF system working in high NA TIRF mode is able to provide an imaging depth shallower than that in a conventional TIRF microscope.

It is worth noting that the penetration depth does not represent an absolute limit on the depth of features in a sample that will be observed [16

16. C. Gell, M. Berndt, J. Enderlein, and S. Diez, “TIRF microscopy evanescent field calibration using tilted fluorescent microtubules,” J. Microsc. 234(1), 38–46 (2009). [CrossRef] [PubMed]

]. In other words, the imaging depth may be deeper than the penetration depth. The penetration depth is artificially defined as the distance at which the intensity of the evanescent field decays to 1/e (corresponding to 37%) of its value at the interface. In addition, the exponential decay of the evanescent field is an oversimplification; precise calibration shows that approximately 90% of the evanescent field is represented by an exponential with a decay consistent with theory whereas the remaining 10% of the field is represented by an exponential with a much longer decay constant and is identified as scattering [17

17. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

]. The source of the scattered light is mainly from multiple reflections at various interfaces and scattering in elements in the objective lens. Moreover fluorescence emitted as a result of excitation by the evanescent field propagates beyond the theoretical penetration depth and elicits further signals from fluorophores, giving an increased imaging depth. In terms of genuine cell samples the local refractive indices of intracellular components are more complicated and heterogeneous; therefore the actual evanescent field may be significantly affected and stronger scattering may occur. For the SITIRF system working in high NA TIRF mode, the higher emission collection efficiency for proximal fluorophores [18

18. J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999). [CrossRef] [PubMed]

] increase the steepness of the decay from pure single exponential function, which is beneficial for the surface confinement in SITIRF microscopy.

1 µm Nile red (excitation/emission peaks: 535/575 nm) fluorescent beads (FluoSphere F-8819, Invitrogen) were used to examine the highly confined surface imaging of the SITIRF microscope. The excitation source was a DPSS laser at 473 nm, and the emission filter was centered at 570 nm with 10 nm spectral bandwidth (570/10 nm, FB570-10, Thorlabs). The images captured in different modes are displayed in Fig. 5
Fig. 5 Images of 1 µm diameter fluorescent beads in (a) epi-fluorescence mode, (b) conventional NA TIRF mode with an illumination NA of 1.45, and (c) high NA TIRF mode with an illumination NA of 2.2. 3D surface plots (d, e, f) of a single bead, as indicated by the dashed box, in different modes are shown on the right-hand panel.
, and the 3D surface plots of a single bead, as marked by the dashed box, are also shown on the right-hand panel. In epi-fluorescence mode the images of these beads, as displayed in Fig. 5(a), 5(d), show a gradually declining intensity distribution from the center to the edge in a 1 µm diameter area. However, in conventional NA TIRF mode (Fig. 5(b), 5(e)) there is an intensity peak on one side where the TIR illumination beam is incident on the fluorescent bead. In this case the 1 µm beads converted evanescent light into propagating comet-shaped light due to relatively deep penetration depth of the evanescent wave [19

19. R. Wannemacher, A. Pack, and M. Quinten, “Resonant absorption and scattering in evanescent fields,” Appl. Phys. B 68(2), 225–232 (1999). [CrossRef]

] and morphology-dependent scattering [20

20. C. Liu, T. Weigel, and G. Schweiger, “Structural resonances in a dielectric sphere on a dielectric surface illuminated by an evanescent wave,” Opt. Commun. 185(4-6), 249–261 (2000). [CrossRef]

]. The similarity of the asymmetric fluorescent emission in Fig. 5(b) and 5(e) with images obtained using an Olympus 60 × 1.45 NA oil immersion objective lens [17

17. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

] confirm that the SITIRF microscope in conventional NA TIRF mode faithfully imitates a conventional TIRF microscope. Figure 5(e), 5(f) shows that in high NA TIRF mode the penetration depth was significantly reduced, theoretically to 19 nm at an ASIL-air interface, so that only a small area where the bead contacted the substrate was illuminated, and then the disruption on the evanescent field was eliminated. As a consequence of this confined illumination, a relatively intensely fluorescent area can be seen at the center of the bead image (Fig. 5(e)) which we regard as the effective imaging area in high NA TIRF mode. Thus the shallower imaging depth in high NA TIRF mode results in a smaller effective imaging area than for epi-fluorescence or conventional NA TIRF modes. From the images shown in Fig. 5 it is clear that the SITIRF microscope is able to provide a stronger confinement of imaging depth than that in either the epi-fluorescence microscope or the conventional TIRF microscope, even on a sample with densely concentrated fluorophores. In Fig. 5(c), 5(f) the residual fluorescence emission in regions outside the effective imaging area arises mainly from excitation by the scattering light at subcritical angles. The amount of this scattering-induced emission remained approximately constant for different illumination NAs as illustrated in Fig. 5, which again is in agreement with the report in [17

17. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

]. The effective imaging depth in high NA TIRF mode can be quantified, based on the geometric model of a fluorescent bead shown in Fig. 6
Fig. 6 Geometric model of a fluorescent bead.
. The imaging depth AB¯can be calculated by:
AB¯=OB¯OC¯2AC¯2=RR2reff2
(7)
where R is the radius of the fluorescent bead and reff is the radius of the effective imaging area. According to the average over 10 beads the imaging depth was 83.8±9.5 nm. This measured imaging depth is greater than theoretically predicted penetration depth mainly due to the high refractive index (nd = 1.592) of the polystyrene bead.

In addition to fluorescent bead images, BFP images, as shown in Fig. 7
Fig. 7 BFP images captured with 1 µm fluorescent bead samples in (a) epi-fluorescence mode, (b) conventional NA TIRF mode with illumination NA of 1.45, and (c) high NA TIRF mode with illumination NA of 2.2. The yellow dashed circles indicate the full NA of the system which is 2.2; the radial positions of the focused beam in each case are indicated by the yellow leader lines.
, were also captured using a 490 nm longpass filter (NT62-983, Edmund Optics) as an emission filter. The illumination NA can be measured from the BFP images as the location of each illumination focusing spot is clearly shown. The radial positions of the focused beam on the BFP in each case are indicated by the yellow leader lines in Fig. 7. According to Eq. (5) we can quantify the illumination NA in conventional NA TIRF mode and high NA TIRF mode as 1.45 and 2.2, respectively, which are consistent with the theoretical values.

Next, we imaged a mixture of 40 nm yellow-green (excitation/emission peak: 505/515 nm) fluorescent beads (FluoSphere F-8780, Invitrogen) and 1 µm Nile red fluorescent beads. In this case the excitation was 473 nm and a 510/10 nm emission filter (FB510-10, Thorlabs) was used. This filter efficiently transmitted the fluorescence wavelengths from the yellow-green beads while eliminating those from the Nile red beads, although a small amount of the Nile red fluorescence did leak through the filter due to the high fluorescence intensity. In Fig. 8
Fig. 8 Images of a mixture of 40 nm and 1 µm fluorescent beads in (a, e) bright-field mode (b, f) epi-fluorescence mode, (c, g) conventional NA TIRF mode with an illumination NA of 1.45, and (d, h) high NA TIRF mode with an illumination NA of 2.2.
we show the images of the mixed samples in (a,e) bright-field mode, (b,f) epi-fluorescence mode, (c,g) conventional NA TIRF mode with illumination NA of 1.45, and (d,h) high NA TIRF mode with illumination NA of 2.2. The 40 nm fluorescent beads maintained a similar level of visibility in these images (except in the bright-field mode due to the weak scattering) because in each case they were very close to the ASIL surface and kept within different illumination depths. However only in high NA TIRF mode did the 1 µm bead became virtually undetectable (Fig. 8(d), 8(h)) because most of the bead was above the penetration depth. Theoretically the volume of the fluorophore in each 1 µm bead that can be detected in high NA mode was less than 28% of that in conventional NA mode, which made the beads nearly undetectable in high NA mode when the similar illumination power density was maintained.

In [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

] it was demonstrated SIF microscopy is a type of wide-field high-resolution microscopy; accordingly, SITIRF microscopy, as a modification of SIF microscopy, should also perform high-resolution on a wide-field basis. In Fig. 8(e)8(h) the fluorescent beads were located at the periphery of the FOV but there is no noticeable degradation of resolution, which proves the SITIRF is able to perform wide-field high-resolution imaging. Quantitatively the resolution performance of the high NA SITIRF system can be estimated from the FWHM of a two-dimensional Gaussian fit to the image of each 40 nm bead as these beads effectively acted as point emitters. The FWHM of a single 40 nm bead image from the average over 10 separate beads was measured as 174 ± 11.5 nm. Considering the actual size of these beads we may use a simple deconvolution method to estimate the PSF [21

21. Q. Wu, G. D. Feke, R. D. Grober, and L. P. Ghislain, “Realization of numerical aperture 2.0 using a gallium phosphide solid immersion lens,” Appl. Phys. Lett. 75(26), 4064–4066 (1999). [CrossRef]

]:
PSF=Δx2observedΔx2bead
(8)
where Δxobserved is the FWHM from the bead image and Δxbead is the actual size of the beads. We conclude that the resolution of the microscope was about 169 nm. The deviation from the theoretical value of 141 nm comes mainly from the difficulty of collecting rays propagating at angles approaching 90°, and the resolution degradation induced by the air gap between the fluorescent beads and the ASIL [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

].

5. Conclusions and discussion

In this paper, we have investigated the application of total internal reflection illumination in wide-field high-resolution solid immersion fluorescence microscopy employing an aplanatic solid immersion lens. This microscopy, referred to as solid immersion total internal reflection fluorescence microscopy, is capable of performing highly confined surface imaging. The ASIL employed in our system was made of a modern crystalline material, ZrO2, which has a higher refractive index and lower dispersion than that of conventional optical glasses. The application of high-index ZrO2 ASIL allowed SITIRF microscopy to achieve higher resolution and shallower imaging depth. In addition, the low dispersion of the material removed the requirement for the narrow band emission filters normally needed to compensate for chromatic aberration, and enabled the use of emission filters with a wider spectral band. The novel design of the experimental SITIRF system enabled us to image the same sample in different illumination NAs, and to evaluate and compare the imaging depths in epi-fluorescence mode, conventional NA TIRF mode and high NA TIRF mode. We demonstrated that highly confined surface imaging can be performed by the SITIRF system in high NA TIRF mode by imaging 1 µm fluorescent beads, a mixture of 40 nm and 1 µm fluorescent beads and also by imaging a GFP-tagged mutant of the cyanobacterium Synechocystis PCC6803. We also demonstrated the SITIRF system can provide high lateral resolution imaging on a wide-field basis.

In contrast to conventional TIRF microscopy, the distinctive advantage of high NA SITIRF microscopy is that highly confined surface imaging enables highly selective imaging on cellular features closely adjacent to the substrate, which makes it more efficient to investigate the structures and dynamics of cell membranes. In addition, high NA SITIRF microscopy considerably eliminates background fluorescence by confining imaging within a very shallow depth, and as a result the resolution and contrast performance are enhanced compared to conventional TIRF microscopy, features that are particularly beneficial for single molecular imaging.

Although we studied highly confined surface imaging using SITIRF microscopy in this work, this technique has great potential for superior performance in many other situations in which conventional TIRF microscopy is applied. SITIRF microscopy can achieve a higher illumination NA, i.e. a larger incident angle of illumination, than that in conventional TIRF microscopy, a feature that leads to the possibility of observing very densely packed cellular components with evanescent wave illumination. For example, lysosomes in EMT6 cells have an index of 1.6 [26

26. J. D. Wilson, W. J. Cottrell, and T. H. Foster, “Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes,” J. Biomed. Opt. 12(1), 014010 (2007). [CrossRef] [PubMed]

] and melanin in cuttlefish has an even higher index of 1.6-1.7 [27

27. I. A. Vitkin, J. Woolsey, B. C. Wilson, and R. R. Anderson, “Optical and thermal characterization of natural (Sepia officinalis) melanin,” Photochem. Photobiol. 59(4), 455–462 (1994). [CrossRef] [PubMed]

]. High NA SITIRF microcopy clearly has the potential to become a powerful tool for studies of the structural dynamics of these components. Another aspect in which SITIRF microscopy may have superior performance exists in axial resolution. The strong z-axis dependence of the excitation intensity in TIRF microscopy has been used to obtain sub-wavelength resolution in the axial direction; for example, TIRF microscopy was employed to observe the axial motion of single secretary granules with an axial resolution of 30 nm [28

28. J. A. Steyer and W. Almers, “Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy,” Biophys. J. 76(4), 2262–2271 (1999). [CrossRef] [PubMed]

] and the complexes on the plasma membrane of living cells to 10 nm axial resolution [29

29. S. Saffarian and T. Kirchhausen, “Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane,” Biophys. J. 94(6), 2333–2342 (2008). [CrossRef] [PubMed]

]. SITIRF microscopy could deliver an even higher axial resolution owing to its higher restraint capability and a finer adjustability of the evanescent field, so these characteristics could be of value for enhancing the performance of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) [30

30. K. Stock, R. Sailer, W. S. L. Strauss, M. Lyttek, R. Steiner, and H. Schneckenburger, “Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device,” J. Microsc. 211(1), 19–29 (2003). [CrossRef] [PubMed]

] as well. The idea of combining high NA SILs with TIR illumination may also be applied in non-fluorescence microscopy for real-time imaging of live cellular events [31

31. G. D. Byrne, M. C. Pitter, J. Zhang, F. H. Falcone, S. Stolnik, and M. G. Somekh, “Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles,” J. Microsc. 231(1), 168–179 (2008). [CrossRef] [PubMed]

].

The major practical problems with the SITIRF microscope as it presently stands include the difficulty of mounting real biological samples on the ASIL, the complexity of ASIL cleaning and realignment procedure following each experiment, and the fixed position of effective FOV. We proposed several methods to solve these problems in [4

4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

], and future improvements of the SITIRF microscope include all the aspects discussed in this article in order to make it suitable for routine use.

Acknowledgments

This material is based upon work supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035. PARC’s role was to provide postdoctoral support for LW to conceive, design and test the instrumentation. The maintenance, growth and initial fluorescence imaging of the cyanobacterial cultures were carried out by DPC and CV, supported by a grant to CNH from the Biotechnology and Biological Sciences Research Council (UK). The authors would like to thank Professor Colin Robinson, Warwick University, UK, for the gift of the PetC3-GFP mutant of Synechocystis and Dr Roman Sobotka, Institute of Microbiology, Trebon, Czech Republic, for help with growing this strain.

References and links

1.

S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57(24), 2615–2616 (1990). [CrossRef]

2.

B. D. Terris, H. J. Mamin, D. Rugar, W. R. Studenmund, and G. S. Kino, “Near-field optical data storage using a solid immersion lens,” Appl. Phys. Lett. 65(4), 388–390 (1994). [CrossRef]

3.

J. Zhang, C. W. See, and M. G. Somekh, “Imaging performance of widefield solid immersion lens microscopy,” Appl. Opt. 46(20), 4202–4208 (2007). [CrossRef] [PubMed]

4.

L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt. 49(31), 6160–6169 (2010). [CrossRef]

5.

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999). [CrossRef]

6.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

7.

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

8.

L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution structured illumination solid immersion fluorescence microscopy,” Opt. Lett. 36(15), 2794–2796 (2011). [CrossRef] [PubMed]

9.

A. L. Stout and D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28(24), 5237–5242 (1989). [CrossRef] [PubMed]

10.

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]

11.

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng. 10(1), 311–338 (2008). [CrossRef] [PubMed]

12.

T. Yanagida, Y. Sako, and S. Minoghchi, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol. 2(3), 168–172 (2000). [CrossRef] [PubMed]

13.

J. Z. Rappoport and S. M. Simon, “Real-time analysis of clathrin-mediated endocytosis during cell migration,” J. Cell Sci. 116(5), 847–855 (2003). [CrossRef] [PubMed]

14.

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng. 13(1), 247–268 (1984). [CrossRef] [PubMed]

15.

P. Török and F. J. Kao, Optical Imaging and Microscopy: Techniques and Advanced Systems,87 of Springer series in optical sciences (Springer, 2007).

16.

C. Gell, M. Berndt, J. Enderlein, and S. Diez, “TIRF microscopy evanescent field calibration using tilted fluorescent microtubules,” J. Microsc. 234(1), 38–46 (2009). [CrossRef] [PubMed]

17.

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

18.

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999). [CrossRef] [PubMed]

19.

R. Wannemacher, A. Pack, and M. Quinten, “Resonant absorption and scattering in evanescent fields,” Appl. Phys. B 68(2), 225–232 (1999). [CrossRef]

20.

C. Liu, T. Weigel, and G. Schweiger, “Structural resonances in a dielectric sphere on a dielectric surface illuminated by an evanescent wave,” Opt. Commun. 185(4-6), 249–261 (2000). [CrossRef]

21.

Q. Wu, G. D. Feke, R. D. Grober, and L. P. Ghislain, “Realization of numerical aperture 2.0 using a gallium phosphide solid immersion lens,” Appl. Phys. Lett. 75(26), 4064–4066 (1999). [CrossRef]

22.

M. Liberton, R. Howard Berg, J. Heuser, R. Roth, and H. B. Pakrasi, “Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803,” Protoplasma 227(2-4), 129–138 (2006). [CrossRef] [PubMed]

23.

D. Schneider, E. Fuhrmann, I. Scholz, W. R. Hess, and P. L. Graumann, “Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes,” BMC Cell Biol. 39(1), 8–39 (2007). [CrossRef] [PubMed]

24.

C. Aldridge, E. Spence, M. A. Kirkilionis, L. Frigerio, and C. Robinson, “Tat-dependent targeting of Rieske iron-sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803,” Mol. Microbiol. 70(1), 140–150 (2008). [CrossRef] [PubMed]

25.

D. Woitzik, J. Weckesser, and U. J. Jurgens, “Isolation and characterization of cell-wall components of the unicellular cyanobacterium Synechococcus sp. PCC 6307,” J. Gen. Microbiol. 134, 619–627 (1988).

26.

J. D. Wilson, W. J. Cottrell, and T. H. Foster, “Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes,” J. Biomed. Opt. 12(1), 014010 (2007). [CrossRef] [PubMed]

27.

I. A. Vitkin, J. Woolsey, B. C. Wilson, and R. R. Anderson, “Optical and thermal characterization of natural (Sepia officinalis) melanin,” Photochem. Photobiol. 59(4), 455–462 (1994). [CrossRef] [PubMed]

28.

J. A. Steyer and W. Almers, “Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy,” Biophys. J. 76(4), 2262–2271 (1999). [CrossRef] [PubMed]

29.

S. Saffarian and T. Kirchhausen, “Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane,” Biophys. J. 94(6), 2333–2342 (2008). [CrossRef] [PubMed]

30.

K. Stock, R. Sailer, W. S. L. Strauss, M. Lyttek, R. Steiner, and H. Schneckenburger, “Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device,” J. Microsc. 211(1), 19–29 (2003). [CrossRef] [PubMed]

31.

G. D. Byrne, M. C. Pitter, J. Zhang, F. H. Falcone, S. Stolnik, and M. G. Somekh, “Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles,” J. Microsc. 231(1), 168–179 (2008). [CrossRef] [PubMed]

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy
(240.0240) Optics at surfaces : Optics at surfaces
(240.6690) Optics at surfaces : Surface waves
(260.6970) Physical optics : Total internal reflection

ToC Category:
Microscopy

History
Original Manuscript: November 23, 2011
Revised Manuscript: January 5, 2012
Manuscript Accepted: January 8, 2012
Published: January 27, 2012

Virtual Issues
Vol. 7, Iss. 3 Virtual Journal for Biomedical Optics

Citation
Lin Wang, Cvetelin Vasilev, Daniel P. Canniffe, Luke R. Wilson, C. Neil Hunter, and Ashley J. Cadby, "Highly confined surface imaging by solid immersion total internal reflection fluorescence microscopy," Opt. Express 20, 3311-3324 (2012)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-20-3-3311


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References

  1. S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett.57(24), 2615–2616 (1990). [CrossRef]
  2. B. D. Terris, H. J. Mamin, D. Rugar, W. R. Studenmund, and G. S. Kino, “Near-field optical data storage using a solid immersion lens,” Appl. Phys. Lett.65(4), 388–390 (1994). [CrossRef]
  3. J. Zhang, C. W. See, and M. G. Somekh, “Imaging performance of widefield solid immersion lens microscopy,” Appl. Opt.46(20), 4202–4208 (2007). [CrossRef] [PubMed]
  4. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution solid immersion fluorescence microscopy applying an aplanatic solid immersion lens,” Appl. Opt.49(31), 6160–6169 (2010). [CrossRef]
  5. R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE3568, 185–196 (1999). [CrossRef]
  6. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE3919, 141–150 (2000). [CrossRef]
  7. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198(2), 82–87 (2000). [CrossRef] [PubMed]
  8. L. Wang, M. C. Pitter, and M. G. Somekh, “Wide-field high-resolution structured illumination solid immersion fluorescence microscopy,” Opt. Lett.36(15), 2794–2796 (2011). [CrossRef] [PubMed]
  9. A. L. Stout and D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt.28(24), 5237–5242 (1989). [CrossRef] [PubMed]
  10. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol.89(1), 141–145 (1981). [CrossRef] [PubMed]
  11. J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng.10(1), 311–338 (2008). [CrossRef] [PubMed]
  12. T. Yanagida, Y. Sako, and S. Minoghchi, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000). [CrossRef] [PubMed]
  13. J. Z. Rappoport and S. M. Simon, “Real-time analysis of clathrin-mediated endocytosis during cell migration,” J. Cell Sci.116(5), 847–855 (2003). [CrossRef] [PubMed]
  14. D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng.13(1), 247–268 (1984). [CrossRef] [PubMed]
  15. P. Török and F. J. Kao, Optical Imaging and Microscopy: Techniques and Advanced Systems,87 of Springer series in optical sciences (Springer, 2007).
  16. C. Gell, M. Berndt, J. Enderlein, and S. Diez, “TIRF microscopy evanescent field calibration using tilted fluorescent microtubules,” J. Microsc.234(1), 38–46 (2009). [CrossRef] [PubMed]
  17. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt.11(1), 014006 (2006). [CrossRef] [PubMed]
  18. J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt.38(4), 724–732 (1999). [CrossRef] [PubMed]
  19. R. Wannemacher, A. Pack, and M. Quinten, “Resonant absorption and scattering in evanescent fields,” Appl. Phys. B68(2), 225–232 (1999). [CrossRef]
  20. C. Liu, T. Weigel, and G. Schweiger, “Structural resonances in a dielectric sphere on a dielectric surface illuminated by an evanescent wave,” Opt. Commun.185(4-6), 249–261 (2000). [CrossRef]
  21. Q. Wu, G. D. Feke, R. D. Grober, and L. P. Ghislain, “Realization of numerical aperture 2.0 using a gallium phosphide solid immersion lens,” Appl. Phys. Lett.75(26), 4064–4066 (1999). [CrossRef]
  22. M. Liberton, R. Howard Berg, J. Heuser, R. Roth, and H. B. Pakrasi, “Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803,” Protoplasma227(2-4), 129–138 (2006). [CrossRef] [PubMed]
  23. D. Schneider, E. Fuhrmann, I. Scholz, W. R. Hess, and P. L. Graumann, “Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes,” BMC Cell Biol.39(1), 8–39 (2007). [CrossRef] [PubMed]
  24. C. Aldridge, E. Spence, M. A. Kirkilionis, L. Frigerio, and C. Robinson, “Tat-dependent targeting of Rieske iron-sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803,” Mol. Microbiol.70(1), 140–150 (2008). [CrossRef] [PubMed]
  25. D. Woitzik, J. Weckesser, and U. J. Jurgens, “Isolation and characterization of cell-wall components of the unicellular cyanobacterium Synechococcus sp. PCC 6307,” J. Gen. Microbiol.134, 619–627 (1988).
  26. J. D. Wilson, W. J. Cottrell, and T. H. Foster, “Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes,” J. Biomed. Opt.12(1), 014010 (2007). [CrossRef] [PubMed]
  27. I. A. Vitkin, J. Woolsey, B. C. Wilson, and R. R. Anderson, “Optical and thermal characterization of natural (Sepia officinalis) melanin,” Photochem. Photobiol.59(4), 455–462 (1994). [CrossRef] [PubMed]
  28. J. A. Steyer and W. Almers, “Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy,” Biophys. J.76(4), 2262–2271 (1999). [CrossRef] [PubMed]
  29. S. Saffarian and T. Kirchhausen, “Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane,” Biophys. J.94(6), 2333–2342 (2008). [CrossRef] [PubMed]
  30. K. Stock, R. Sailer, W. S. L. Strauss, M. Lyttek, R. Steiner, and H. Schneckenburger, “Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device,” J. Microsc.211(1), 19–29 (2003). [CrossRef] [PubMed]
  31. G. D. Byrne, M. C. Pitter, J. Zhang, F. H. Falcone, S. Stolnik, and M. G. Somekh, “Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles,” J. Microsc.231(1), 168–179 (2008). [CrossRef] [PubMed]

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