Tomographic Phase Microscopy (TPM) - Biomedical Optical Phase Microscopy and Nanoscopy - page 239

600 m. Light passing through the sample is collected by an oil-immersion objective lens

(Olympus UPLSAPO 100XO, 1.4 NA), and an achromatic doublet tube lens ( f 5 200 mm) was
used to focus an image of the sample onto the camera with magnification M 5 250. The
reference laser beam is enlarged by a 10 3 beam expander (L2, L3) and combined with the
sample beam through a beamsplitter. The resulting interference pattern is captured at 10-bit
resolution by a high-speed CMOS camera (Photron Fastcam APX RS, 512 3 512 pixels).
A mercury arc lamp, LED illuminator, dichroic mirror, optical filters, and CCD camera
(Roper CoolSnap HQ) are also integrated into the setup for correlated brightfield and
fluorescent imaging. The galvanometer is driven by a symmetric triangle wave with amplitude
corresponding to 6 60 at the sample and frequency of 15 Hz. A total of 150 images are
acquired during each galvanometer sweep. Irradiances at the detector plane are B 10

μ

W/cm 2 μ

for both the sample and reference fields; camera exposure times were typically B 20 μ s.

To obtain angle-dependent quantitative phase images, a fringe pattern demodulation
technique [29,30] is used. First, the Fourier transform of the raw image is calculated;
it contains peaks centered at 0 and 6 !

θ ; where !

is the spatial frequency of the fringe
pattern, equal to the difference between sample and reference wave vectors at the image
plane. The Fourier components are then shifted by 2 !
θ

θ

such that the 1 !
θ

peak is translated
to 0. A 2D Hanning low-pass filter is applied to select only this central component.
Applying the inverse Fourier transform then gives a complex-valued function Z θ ( x , y ), from
which the phase image is calculated by φ θ ( x , y ) 5 Arg Z θ ( x , y ).

L1

GM

θ

C

S

OBJ

BS

TL CMOS

HeNe BS

DBS

CCD

TL

L2 BE L3

Figure 12.10
Spatial modulation tomography setup; HeNe, helium neon laser. In sample path (red)—BS,

beamsplitter; GM, galvanometer-controlled mirror; C, condenser lens; , beam tilt angle;

S, sample; OBJ, objective lens; and DBS, dichroic beamsplitter. In reference path (blue)—BE,
beam expander; TL, tube lenses; CMOS, camera; L1

θ

L3, lenses; CCD, camera for brightfield and
fluorescent imaging (light path shown by dotted line); and not shown, illuminators and filters for
brightfield and fluorescent imaging [25] .