Correlated Microscope
Table 1: Optical components for ELVIS. The numbered parts are shown in the schematic in Figure 2. Illumination Collimator
Filters DHM 405 nm
diode laser (Thorlabs S1FC405) (1a)
100mm focal length achromat; 1” diameter Newport Optics PAC052AR.13 (2a)
405×10 nm bandpass filter (3)
Beamsplitter Objectives
BS1: 425 nm shortpass dichroic
beamsplitter 25×36mm (Thorlabs DMSP425R)
Pair (science beam/ reference beam): 10mm focal length achromats; 6.25mm diameter (masked to 5 mm)
(Edmund
FLFM 470 nm LED (Thorlabs M470L3) (1b)
20.1mm focal length; 1” diameter (Thorlabs ACL2520U- A)(2b)
Excitation: 469±35 nm bandpass (Thorlabs MF469-35) (4)
Emission: 500 nm longpass (Thorlabs FELH0500) (5)
BS2: 490 nm shortpass dichroic; 25×36mm (Thorlabs DMSP490/R
Optics 47689) Same as
DHM;only use one of the objectives (6)
200 mm focal length achromat; 2” diameter
(Thorlabs AC508- 200-A) (7b) Lenslet array 3.75mm focal length and 125 μm pitch (RPC Photonics RPC125-f30) (8) 2:1 Telecentric optical relay (Opto Engineering TC23-016) (9)
to demonstrate practical applications of the combined DHM/ FLFM system.
Materials and Methods Te design of the DHM side of the instrument has been
described in detail [4], with small differences in component selection for the combined DHM/FLFM instrument (Table 1). Te DHM/FLFM system was developed with the goal of using a single objective, or a set of objectives, for both modes of the instrument, but with otherwise independent optics. In prin- ciple the DHM light source can be used for the FLFM illumi- nation, but in practice there are advantages, described below, to using separate illumination capability for both modes. Note that one of our goals was to make use of relatively simple and inexpensive objective lenses to reduce the cost, complexity, and number of optical surfaces involved. Figure 2 shows a sche- matic and photo of the instrument, illustrating the instrument elements listed in Table 1.
Results ELVIS standardized sample testing. Amplitude resolution
laterally (x, y) and axially (z), depth of field, and phase sensitiv- ity were all measured using U.S. Air Force (USAF) test targets [Edmund Optics SKU 58-198]. DHM-mode amplitude recon- structions without noise subtraction showed lateral resolution better than 0.9 µm (Figure 3a). No measurable loss of resolu- tion in the reconstructed images was seen at a range of 900 µm in z. Since this exceeded the depth of our sample chambers,
2020 May •
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Same as DHM or RGB camera of choice (10b)
Tube lens
150 mm focal length achromat; 2” diameter
(Newport Optics PAC086AR.13) (7a)
Camera
2464×2056 pixels
(windowed to 2048×2048 in operation) 3.45×3.45 μm pixels
(AlliedVision GT2460) (10a)
we did not measure depth of field farther. A measure of axial resolution was obtained by translating the micrometer stage a known distance in z and comparing this known distance with the position of best reconstructed focus. Tese values should differ by the square of the magnification of the system (approx- imately 218). A best fit to these values gives a slope of 228 with fit residuals of 9.6 µm at the sample, which corresponds to 4.4 µm axial resolution (Figure 3b). A phase target consisting of patterns of known thickness
was imaged, and the difference between the averages of a patch within the largest square and a similar patch just outside was measured (Figure 3c). Te graph in Figure 3d plots the mea- sured phase delay in nm versus the actual thickness in nm. Te best fit slope is 0.52 +/− 0.02 (with a small offset). Te slope corresponds to n-1, where n is the index of refraction of the phase material, corresponding to the manufacturer’s value. Te residuals are 4 nm, or 1% of a wave. On the FLFM side, the field of view was measured to be
790×660 µm, which exceeded that of the DHM. Te two fields of view overlapped to a large extent, but not completely. Fig- ures 3e and 3f show raw and reconstructed USAF target images obtained with the FLFM that resolved line group 6,5, implying a resolution aſter reconstruction of 4.9 µm. Te lateral resolution degraded with axial offset, leading to a depth of field of 150 µm with a resolution of 5.5 µm, and 300 µm for 7.0 µm resolution. Axial resolution was also measured using 100 nm SiO2
beads (Polysciences,
Inc. 24041-10). Amplitude and phase 21
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