Display Settings:

Items per page

Results: 6

1.
Fig. 6

Fig. 6. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

SIFM image of microvasculature in a mouse heart. The image represents a 500 x 500 x 60 µm volume of tissue, starting at 15 µm below the tissue surface. (A) The depth of the vessels is color-coded: the deepest layers are displayed in violet and the top layers are displayed in red. (B) A movie that shows a three-dimensional rendering of the data is available online ( Media 2).

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.
2.
Fig. 3

Fig. 3. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

Schematic representation of the SIFM microscope. Excitation light from a fiber coupled laser is sent through a wavelength division multiplexer (wdm) and a collimator (col) before passing through a phase plate (PP). The beam is sent into the microscope (IX71) via a home-built galvanometric x/y-scanner (xy) and is focused onto the sample by the objective (obj). The fluorescence is collected by the same objective and follows the same light path in the opposite direction to the wdm where it is sent to a home-built spectrometer that detects the interference spectra.

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.
3.
Fig. 5

Fig. 5. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

Comparison of SIFM and confocal microscopy on a three-dimensional distribution of fluorescent microspheres. (a) SIFM intensity image (100 × 100 µm). The red square marks the area that was compared to a standard confocal stack. (b) SIFM phase image. (c) Representative slice from the SIFM 3D reconstruction. (d) Corresponding slice from the confocal stack. (e) SIFM and confocal slices overlapped. SIFM data displayed in green and confocal in red. Yellow indicates the overlap of both data sets. A video that shows the overlap of the data sets for the whole volume is available online ( Media1).

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.
4.
Fig. 2

Fig. 2. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

Schematic representation of the ray matrix model. The rays vinemitted by a source at a defocus distance δ from the focal plane of the objective L0 are traced by multiplication with the free space propagation matrices Si and the lens matrices Li. From the position vout and the angle θout we can calculate the radius of curvature R of the wave front at the phase plate. This allows us to calculate the optical path delay opd(y,δ) which we define as the difference in optical path to the phase plate between a ray intersecting the phase plate at y and the chief ray for which y = 0. Averaging of opd(y,δ) over the inner and outer sections of the phase plate now yields Δopl(δ)the extra path length difference between the field passing through the center and the field passing through the edge of the plate that is caused by the wave front curvature (see Fig. 1).

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.
5.
Fig. 4

Fig. 4. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

Phase and SNR. (a) Dependence of the phase of the self-interference spectrum on the axial position of a thin homogenous fluorescent layer with respect to the focal plane of the objective. Each datapoint is the mean value from 1024 spectra. The error bars indicate ± 1 standard deviation. The imaging NA for this measurement was 0.086 (using the approximate definition for the NA of Gaussian beams:NA ≈2λ / πw0) and the Rayleigh lengthzRwas 128 µm. A linear fit of the central section between −2 and 2zRyields a slope dϕ/dz = −1.31 rad/zR. A simple ray optics analysis of the system predicts a slope of −1.33 rad/zRindicated by the solid gray line. (b) Dependence of the standard deviation of a phase measurement on the signal to noise ratio. Here the SNR was varied by scanning the fluorescent layer through the focus of the objective. The dots are the measured standard deviations of 1024 phase measurements. The solid line is the theoretical curve .

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.
6.
Fig. 1

Fig. 1. From: Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

Principle of SIFM.Light from a fluorescent source in the sample is collimated by the objective lens and passes through a phase plate. The phase plate consists of an inner and an outer ring of different optical thickness, introducing two alternative optical paths. The wave front before and after the phase plate is drawn as a solid red line. The light passing through the thicker outer ring of the phase plate is retarded with respect to the light passing through the hole in the center. The light is focused on a single mode optical fiber, acting as a pinhole. The interference due to the optical path difference leads to a modulation on the detected fluorescence spectrum: for certain wavelengths the interference is destructive while for others it is constructive (top graph). The period of the spectral modulation is determined by the thickness of the plate. The modulation depth depends on the ratio of the integrated amplitude of the source field over the central disk of the phase plate and the outer ring, respectively. When the source is exactly in focus (δ = 0 µm) the wavefronts are flat. When the source is out of focus (δ = 100 µm) the wavefronts are curved (shown exaggerated). This leads to a small extra path length difference and to a shift in the phase of the self-interference spectrum (bottom graph). The phase therefore directly encodes the axial position of the fluorescent source.

Mattijs de Groot, et al. Opt Express. 2012 July 2;20(14):15253-15262.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk