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Dear Martin,
To add what's been said already... I've observed this behaviour sometimes over the years as well and have never been quite able to explain it. Using home-built and commercial TIRF systems, I have seen cases where the sample looks as expected both under EPI and TIRF illumination conditions, and only days later the same preparation does not. There have even been cases where it looks great in one FOV of the sample, but then I move around to another section, and it does not. It those cases, the cells usually contained very bright and large vesicles that most likely have a high refractive index.
What I have found empirically is that samples that are overly confluent tend to have "non-TIRF" or widefield components in the image, and this most likely is due to sideways scatter of the illumination beam through the sample. Evanescent waves are horizontally traveling light waves that propagate sideways along the coverslip after all. If they scatter, they can become non-evanescent and excite things beyond the TIRF illumination volume. The previous email responses called this "light piping". I think another term for this is "frustrated TIR".
There are a few papers that have discussed the scattering issue:
Mattheyses, A.L. and D. Axelrod, Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence. Journal of Biomedical Optics, 2006. 11(1).
"The intensity of the evanescent field may be sensed by a fluorescent probe. A challenge in measuring the evanescent field is to avoid disrupting it. To determine the z profile directly, it is desirable to use a single probe large or bright enough to track. However, if such a probe has different refractive index than the surrounding liquid, the material will scatter light. A spherical probe un- matched in refractive index converts evanescent light into propagating beacons outside the sphere. This can be seen readily in fluorescent solution Fig. 1a. The intensity pattern inside a sphere can be even more complicated. However, if the index of refraction of the surrounding media and the probe object are equal, there will be no dielectric boundary and the evanescent field will remain pure and unscattered. Index matching places a significant restriction on the types of materials appropriate for the probe object. The index of refraction of the sample must be lower than that of the glass coverslip to satisfy the physical requirements for total internal reflection.
Schapper, F., J.T. Goncalves, and M. Oheim, Fluorescence imaging with two-photon evanescent wave excitation. European Biophysics Journal with Biophysics Letters, 2003. 32(7): p. 635-643.
"EW imaging of biological samples poses some unique experimental problems associated with the presence of scattered photons. Scattering creates non-vanishing real components of the wave vector in the direction of the surface normal, causing excitation photons to ‘‘leak’’ into deeper sample regions and the fluorescence signal to contain both near- and far-field components (Oheim and Stuhmer 2000; Loerke et al. 2002; Rohrbach 2000)."
Loerke, D., W. Stuhmer, and M. Oheim, Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation. Journal of Neuroscience Methods, 2002. 119(1): p. 65-73.
"Cells do not make flat contact with the surface but display adhesion sites and ruffles (Truskey et al., 1992; Burmeister et al., 1994) making cytosolic field penetration variable. Together with intracellular refractive-index boundaries, these focal adhesion sites result in light scattering (Oheim and Stu ̈hmer, 2000; Rohrbach, 2000), causing a spread of fluorescence excitation in the direction of beam propagation."
Brunstein, M., et al., Eliminating unwanted far-field excitation in objective-type TIRF. Part I. Identifying sources of nonevanescent excitation light. Biophysical Journal, 2014. 106(5): p. 1020-1032.
"ABSTRACT Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanes- cence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and ren- ders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accu- rately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2)."
Brunstein, M., K. Herault, and M. Oheim, Eliminating unwanted far-field excitation in objective-type tire part ii. Combined evanescent-wave excitation and supercritical-angle fluorescence detection improves optical sectioning. Biophysical Journal, 2014. 106(5): p. 1044-1056.
"ABSTRACT Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objec- tive than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar micro- scopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the con- ditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane."
Cheers,
John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca
On 2015-08-01, at 2:38 PM, Jeff Carmichael wrote:
> *****
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>
> Martin,
>
> Some of this could also be scattered light from the TIRF laser, "piping" in
> along the beads...
>
> Independent of other optical performance, the degree of flatness of the
> dichroic surface influences performance in TIRF, such that "flatter"
> dichroics largely eliminate some artifacts like interference patterns in the
> image or specular reflections or detection of other stray light. The level
> of surface flatness that we've arrived at to be sufficiently flat (in most
> cases) for a dichroic surface (measured after mounting into a microscope
> filter cube) is approx: =< 0.5 waves/inch Peak-to-Valley RWD (Reflected
> Wavefront Distortion) for 2mm-thick dichroics which we use as the "standard"
> TIRF configuration. We also offer =< 0.25 Peak-to-Valley waves/inch
> Peak-to-Valley RWD
>
> We've found that this alone (changing to a cube in the microscope
> fluorescence turret which housed a flatter dichroic)) results in better
> signal/noise, and have some data indicating that this is at least partly
> because fewer TIRF laser photons are propagated directly into the sample and
> are instead properly reflected - maybe because of an improved surface
> flatness profile. We also have much anecdotal data and customer feedback
> which is consistent with greater signal/noise and lack of problematic
> artifacts, and limiting of fluorescence to the TIRF zone.
>
> This is also at least partially due to the increased levels of TIRF laser
> attenuation that our fully-assembled TIRF cubes provide. At the risk of
> being crass, an example albeit commercial, is described below of improving
> the TIRF performance of an advanced and appropriately configured microscope
> in TIRF mode detecting fluorescent beads, as you describe. In this case,
> the beads are coating the surface of a coverglass and also suspended above
> those beads, diluted in a layer of agarose on the surface of the coverglass.
> https://www.chroma.com/sites/default/files/TIRF.pdf
>
> With one dichroic (good quality, sputtered coating, fused silica, basic
> laser quality flatness), beads were visible several microns into the sample
> in TIRF mode in a 3D data set. When we simply changed cubes,
> reset/reconfirmed TIRF beyond the critical angle as before, these deep
> objects were not detected in an otherwise identical data set. SIM data
> shows that the fluorescent objects in the first (beyond the TIRF zone) case
> correlate to real objects suspended in the agarose, and undetected in the
> second case. Neither changing cubes back and forth, nor any particular
> order of imaging had any detectable independent effect.
>
> Best,
> Jeff
>
>
> Jeff Carmichael
> Technical and Product Marketing Mgr.
> [log in to unmask]
>
> Chroma Technology Corp.
> an employee owned company
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>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of Andreas Bruckbauer
> Sent: Saturday, August 01, 2015 2:19 PM
> To: [log in to unmask]
> Subject: Re: TIRF question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> Martin,
> I would think that the refractive index of the bead is similar to glass and
> therefore you don't have TIRF conditions locally where the bead touches the
> coverslip. But this would also imply that the beads are very close together
> in the aggregate and pipe the light efficiently. Check what material they
> are, polystyrene has a refractive index of 1.6 at 500 nm, so this might lead
> to the effect. I usually see only beads on the coverslip, but sometimes very
> bright features of cells are visible because they are still excited in the
> exponential decaying field.
> Best wishes
> Andreas
>
>
> -----Original Message-----
> From: "Martin Wessendorf" <[log in to unmask]>
> Sent: 01/08/2015 17:08
> To: "[log in to unmask]" <[log in to unmask]>
> Subject: TIRF question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List--
>
> I'm a newbie to TIRF microscopy and have a question. Using our Zeiss TIRF
> 'scope, I can often clearly image structures in my biological prep (cultured
> cells) that are in a deeper focal plane than the cover slip.
> This is true even using fairly high TIRF angles (e.g. 80 degrees).
>
> Using TIRF, I would expect that only one focal plane would be visible:
> the plane in contact with the cover slip. This mostly appears to be the
> case when I'm imaging a dilution of fluorescent beads: using epi
> illumination, I can see beads throughout the thickness of the sample whereas
> using TIRF illumination, I can only see those beads that are stuck to the
> cover slip, or that transiently diffuse close enough to the coverslip that
> they flicker into appearance for a moment or two.
> However, if clumps of beads have aggregated and are in contact with the
> cover slip, I can image these beyond the plane of the cover slip--sometimes
> a micron or more beyond.
>
> Anyone got an explanation for how this occurs? Light piping? Are some
> structures just so bright that the evanescent wave is able to excite them,
> even at that distance?
>
> Thanks!
>
> Martin Wessendorf
>
> --
> Martin Wessendorf, Ph.D. office: (612) 626-0145
> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
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