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Digital Holography Microscope

Motorized - Single-Shot Full Resolution

Model : HO-DHM-UT01-FA

    DHM type :     Transmission, Upright / Inverted Dual mode operation
    Interferometer configuration :     Balanced, afocal interferometer
    Optical components :     Optical components used in interferometer (mirrors, beamsplitters)
    Measurement mode :     Single wavelength, single-shot off-axis operation (without Phase Shifting)
    Source :     Diode laser
    Source wavelength :     650 nm
    Source power :     5 mW
    Object beam microscope objectives :     Plan 40X, NA = 0.75    and    20X, NA = 0.50
    Reference beam microscope objectives :     Plan 40X, NA = 0.75    and    20X, NA = 0.50
    Axial depth profiling accuracy :     = 100 nm Lateral resolution : = 1 μm (Full diffraction limited resolution)
    Field of view :     0.180 x 0.120 mm 2 or more
    LED - Laser selection :     Motorized shutter for switching between bright-field and phase modes
    Phase reconstruction :     Through software
    Optical system :     Infinity corrected (200mm or appropriate tube lens)
    Illumination :     Transmission type
    Illumination system :     High bright white LED
    Intensity adjustable Nosepiece :     Quadruple, motorized rotating turret
    Viewing head :     Siedentopf Trinocular head, 30º inclination, 48 - 75mm adjustment
    Eyepiece :     10X wide field (FN20), diopter adjustable, High eye relief
    Microscope Objectives :     1. Plan 4X, NA = 0.13
        2. Plan 10X, NA = 0.30
        3. Plan 20X, NA = 0.50
        4. Plan 40X, NA = 0.75
    Focusing :     Motorized Z stage with autofocusing
    Sample Stage :     Motorized XY stage with Joy stick as well as pc controller with specimen holder
    XY travel :     75mm x 50 mm or more
    Power Input :     230V 50Hz
Camera Specifications

    Sensor type :     CMOS Color
    Shutter :     Global shutter
    Resolution :     3 Mpix or more
    Pixel size :     3.5 μm or smaller
    Frame rate :     120 fps or better
    ADC :     12 bit
    Colour depth :     12 bit
    Optical sensor class :     1 / 1.8”
    Interface connector :     USB 3.0
    Subsampling factor :     2, 4, 8
    Mount :     C-mount
    Minimum exposure time :     0.05 ms or less
    Software :     Software interface for image capture and standard image processing tasks. Will be integrated with phase reconstruction software

Digital holographic microscopy is an emerging modality that offers capability of quantitative phase imaging (QPI) of transparent unstained cells in their most natural state. While phasesensitive imaging methodologies such as dark-field, phase contrast and differential interference contrast are known for several decades, they cannot provide quantitative phase information. DHM can achieve this by use of interferometric imaging concept. A schematic DHM system is shown in Fig. 1:

Fig.1: Schematic of a balanced DHM system that works on the interferometric imaging principle. The phase image is obtained by digital processing of the interference signal recorded using an array sensor.

SF : Spatial filter, BS : Beamsplitter, O : Object beam, R : Reference beam, MO1 / MO2 : Microscope Objectives

When a collimated laser beam passes through a transparent cell sample, typically very little light is absorbed. The laser wavefront however gets distorted due to the phase delay seen by the beam at each (x, y) location (see Fig. 2). The phase Φ (x, y) of the beam after passing through the sample may be described as:

Φ  (x,y)  =  ( 2π / λ )  dz  n (x, y, z)

Here λ is the wavelength of laser used and n (x, y, z) stands for the relative refractive index of the cell at location (x, y, z) relative to the surrounding medium. Clearly a strong phase signal is detected if there is large index difference between the cell and the surrounding medium. If the index of the cell is uniform over a region, the phase function can be approximately associated with the height map profile of the cell, thus giving a 3D perspective of the cell. Instead of employing external contrast agents, the DHM thus uses the natural refractive index contrast of the cells for imaging purpose. Refractive index is a property related to chemical composition and therefore a sensitive phase imaging system can have several applications in basic Bio-sciences and diagnostics.

Fig.2: Illustration of phase delay of a collimated laser beam wavefront as it passes through a transparent cell sample. The phase delay is due to differential optical path difference through different parts of the cell sample.

The Fourier Transform Method is a popular choice for processing of single shot interferometric imaging data. However this method is known to have poor spatial resolution which is well below the diffraction-limited resolution that the microscopic system can achieve. The IIT Delhi technology used in this product uses a novel constrained optimization approach to recovery of full-resolution phase images as illustrated in where bright-field and phase images of a cervical Fig. 3 cell are shown.

Fig.3: Illustration of phase images of red blood cells and patient cervical cells using Digital Holographic Microscopy system (a) Brightfield images, (b) phase images reconstructed using the traditional Fourier transform method, (c) high resolution phase images obtained using novel single shot phase imaging technology developed at IIT Delhi. The color coding in (b) and (c) indicates the phase map (approximately height map) of the cell.

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