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                            University of South Florida
Scholar Commons
Structured light for three-dimensional microscopy
	Leo G. Krzewina
		Scholar Commons Citation
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University of South Florida
Scholar Commons

Graduate Theses and Dissertations Graduate School


Structured light for three-dimensional microscopy
Leo G. Krzewina
University of South Florida

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Krzewina, Leo G., "Structured light for three-dimensional microscopy" (2006). Graduate Theses and Dissertations.
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Page 72

eliminate moving parts, the spatial light modulator (SLM) has also been employed [9,

10]. While longitudinal chromatic aberration (LCA) is generally disadvantageous, when

a known amount of LCA is introduced into the CSM setup it provides depth information

via color, making the axial scan unnecessary [73].

In structured illumination microscopy (SIM) [13] an optical section, or focused

slice, is obtained at any axial position by interjecting a moving linear sinusoidal grid into

the illumination path and capturing three sequential images corresponding to grid phases

incrementally offset by one-third of a spatial period. Simple image processing combines

the three images into a focused section, and the autofocus image is composed from the

brightest points of the images resulting from an axial scan. Lower than expected

sectioning strength in a recent SIM experiment using a very fast smart pixel detector

array under white light illumination was attributed to LCA [69] but not discussed in

detail. We observed similar reduced sectioning strength in the parallel SIM method of

color structured illumination microscopy (CSIM) in which the three phase offsets of SIM

are substituted by the red, green, and blue channels of the CCD camera [65]. Our present

goal is to better understand the limitations imposed by LCA.

Returning to other three-dimensional imaging methods, the fastest way to extend

the depth of field is to eliminate scanning entirely. In digital holography, the intensity

image and phase-unwrapped surface profile are successfully combined to produce the

extended focused image from a single exposure [37]. Wavefront coding is another

promising, single acquisition approach that employs a pupil mask and computer

processing to deconvolve a nominally blurred image to visualize the extended depth of

field [74]. Here we concentrate on the effects of chromatic aberration in SIM. However,


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CA should be considered in any polychromatic experiment, and some of the results

presented are general enough to be applied elsewhere.

6.2. Experimental Setup

The experimental setup is shown in Fig. 6.1. Incoherent light from a high-

intensity white LED is directed toward a grid in a Kohler illumination arrangement. The

structured light passes through a non-polarizing beam splitter to be focused by a

microscope objective (10 NA tube length corrected) onto the object. / 0.25×

Figure 6.1: Experimental setup: L: collimating lens; GRID: spatial light modulator or 35 mm slide of color grid
pattern; BS: beam splitter; MO: microscope objective (10 / 0.25× NA); S: sample.

Light scattered by the object passes back through the microscope objective and

beam splitter, and is imaged onto the camera, a Sony XCD-X710CR color camera with a

Bayer filter. The capture area is 800 600× pixels of size 4.65 4.65 mµ× 2. The image is

processed on a standard personal computer. The grid and camera are both conjugate to


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