15,802 research outputs found
Confocal microscopy
Chapter focusing on confocal microscopy. A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy
Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index
Accurate distance measurement in 3D confocal microscopy is important for
quantitative analysis, volume visualization and image restoration. However,
axial distances can be distorted by both the point spread function and by a
refractive-index mismatch between the sample and immersion liquid, which are
difficult to separate. Additionally, accurate calibration of the axial
distances in confocal microscopy remains cumbersome, although several high-end
methods exist. In this paper we present two methods to calibrate axial
distances in 3D confocal microscopy that are both accurate and easily
implemented. With these methods, we measured axial scaling factors as a
function of refractive-index mismatch for high-aperture confocal microscopy
imaging. We found that our scaling factors are almost completely linearly
dependent on refractive index and that they were in good agreement with
theoretical predictions that take the full vectorial properties of light into
account. There was however a strong deviation with the theoretical predictions
using (high-angle) geometrical optics, which predict much lower scaling
factors. As an illustration, we measured the point-spread-function of a
point-scanning confocal microscope and showed that an index-matched,
micron-sized spherical object is still significantly elongated due to this PSF,
which confirms that single micron-sized spheres are not well suited to
determine accurate axial calibration nor axial scaling.Comment: 8 pages, 5 figure
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Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.
We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations
Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells
Standing-wave excitation of fluorescence is highly desirable in optical
microscopy because it improves the axial resolution. We demonstrate here that
multiplanar excitation of fluorescence by a standing wave can be produced in a
single-spot laser scanning microscope by placing a plane reflector close to the
specimen. We report that the relative intensities in each plane of excitation
depend on the Stokes shift of the fluorochrome. We show by the use of dyes
specific for the cell membrane how standing-wave excitation can be exploited to
generate precise contour maps of the surface membrane of red blood cells, with
an axial resolution of ~90 nm. The method, which requires only the addition of
a plane mirror to an existing confocal laser scanning microscope, may well
prove useful in studying diseases which involve the red cell membrane, such as
malaria.Comment: 15 pages, 4 figures; changed the discussion of narrow-band detected
fringes (Fig. 3) to describe the phenomenon as a moire pattern between the
excitation and emission standing-wave fields, rather than a beats pattern;
added DiI(5)-labelled red blood cell in Fig. 4 to show that standing-wave
fringes are present even when the dye excitation wavelength is outside the
haemoglobin absorption ban
Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates
Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format
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