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Digital Holography Microscope - Inverted

Model No: HO-DHM-IT01

HO-DHM-IT01 is an Inverted type Digital Holography Microscope which can be used for the quantitative phase imaging for live cells. It is a transmission type inverted digital holography microscope with balanced afocal configuration that uses infinity corrected Plan achromatic objectives in both reference and object beams. A common tube lens is provided for achieving afocal imaging configuration with minimal aberrations. A single shot high resolution method patented by IIT Delhi is used for the phase image reconstruction. A 650nm diode laser is used for the construction of holographic image. A high bright white LED illuminated brightfield observation with binocular viewing is also integrated in the system. Since users may not be familiar with phase images, the brightfield observation can be used first to focus the cell sample as desired, followed by recording the phase image using laser illumination. The nosepiece in both object beam and reference beam is equipped with a sliding mechanism for holding two microscope objectives at a time. The user can easily switch the objectives manually in both object beam and reference beam simultaneously by rotating a knob.

A manually operated mechanical stage with 126 * 78 mm travel is equipped with different types of holders for holding different types of sample slides / dishes.

A manually operated shutter with source selector switch is provided near the eyepiece for the safety of eyes by blocking the laser light to the eyepiece. Another automatic shutter is positioned in front of the laser to block the laser beam when the system is used for brightfield observation.Once the desired area for phase imaging is selected in brightfield mode the user can close the manual shutter and the system will be ready for QPI. A complete user-friendly software (Digital HM_V02) with laptop is provided with the system for the recording and reconstruction of the images.

Digital holographic microscope with user specified high end microscope objectives (Plan apochromatic, Plan fluorite, Extra-long WD etc.) may also be made available upon special requests.

    Unique single shot high resolution and accurate quantitative imaging capability (patented technology).

    No phase shifting required thus removing the need for piezo stages.

    Quantitative phase imaging without staining of cells.

    Quantitative phase imaging of live cells.

    Full diffraction limited resolution performance.

    Digital holography as well as transmitted LED brightfield observation.
    Quantitative study of live cells.

    Imaging of various cell types, including SKOV-3 ovarian cancer cells, fibroblast cells, testate amoeba, diatom skeletons, and red blood cells.

    3D imaging and statistics of RBCs in multiple deformation states.

    Evolution of Physiological parameters of different cells.

    Non-destructive analysis of Living cells without any contrast agent: Cell number, confluence, proliferation, cell death, migration and viability.

    Visualization of drug induced morphology changes.

    Visualization of stained or unstained / unprocessed cells.

    Toxicity studies

    Diagnosis of diabetes, and to evaluate long-term glycemic controls in patients with diabetes.

    Resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

    Screening and diagnostics material science.

Theoretical approach

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 phase-sensitive 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.

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.


    DHM type : Transmission, Inverted
    DHM configuration : Balanced, afocal
    Measurement mode : Single wavelength
    Source : Diode laser
    Source wavelength : 650 nm
    Source power : 5 mW
    Object beam microscope objective : Plan Achromatic (40X, 0.65 NA),  Plan Achromatic (20X, 0.40 NA)
    Reference beam microscope objective : Plan Achromatic (40X, 0.65 NA),  Plan Achromatic (20X, 0.40 NA)
    Axial depth profiling accuracy : ≤ 50 nm
    Lateral resolution : ≤ 1 μm
    Camera Field of view : 0.177 x 0.133 mm2
Camera Specs

    Sensor type : CMOS Color
    Shutter : Global shutter
    Pixel class : 3.17 Mpix
    Resolution : 2056 x 1542 Pixels
    Aspect ratio : 4 : 3
    Pixel size : 3.45 μm
    ADC : 12 bit
    Optical size : 7.093 mm x 5.320 mm   (8.87 mm diagonal)
    Optical class : 1 / 1.8”
    Manufacturer : Sony
    Gain (master / RGB) : 24x / 4x
    Frame rate free run mode : 123 fps
    Frame rate trigger (continuous) : 123 fps
    Frame rate trigger (maximum) : 134 fps
    Exposure time (min - max) : 0.018 ms - 999 ms
    Long exposure (maximum) : 30000 ms
    Interface connector : USB 3.0 micro-B
    Software : Digital HM_V02
    Phase image reconstruction : Patented single shot high resolution method
Bright Field Observation

    Optical system : Infinity corrected (200 mm tube lens)
    Observation method : Brightfiled
    Brightfield configuration : Inverted
    Illumination : Transmitted
    Illumination system : High bright white LED, Intensity adjustable
    Nosepiece : Sliding mechanism for Two Objectives
    Microscope Objectives : 1. Plan Achromatic 20X, NA 0.40, 1mm FOV
    2. Plan Achromatic 40X, NA 0.65, 0.5mm FOV
    Viewing head : Sidentopf Binocular head, 45º inclination, 48 - 75mm IP adjustment
    Eyepiece : 10X wide field (FN20), diopter adjustable, High eye relief
    Focusing : Coaxial coarse / fine focusing, fine 0.2mm / rotation
    Stage : Mechanical stage = 126mm x 78mm, co-axial dropdown knob
    Stage Holders : Petridish holder 35mm, Petridish holder 100mm, Glass slide holder, Universal holder for terasaki plate holder, glass slide, dia.35-65 dish and hemocytometer
    Power : 230V 50Hz

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