Digital Holographic Microscope for Surface Analysis

DHM Measurement principle

The DHM™ instrument family generates, in real-time, high resolution 3D digital images of a sample surface using the principle of digital holography. Holograms are generated by illuminating the sample with a coherent laser beam, combining a reference wave with the wave received from a specimen

Figure 1: Reflective mode DHM Single wavelength configuration

Holograms are recorded by a CCD camera and transmitted to a computer for real-time numerical reconstruction. DHM™ Koala software procedures allow computation, from a single hologram acquired in just a few microseconds, of the complete wavefront emanating from an object, providing:-

Intensity image: shows the same contrast as with classical optical microscopy.

Phase image: provides quantitative data, used for accurate and stable measurements. the phase image reveals directly the surface topography with a sub-nanometre vertical resolution.

Holograms are acquired in single acquisition, so the instrument is insensitive to vibrations.
                                                       
Dual Wavelength DHM
The standard DHM setup use a single laser wavelength to generate images. By using a second laser wavelength it is possible to generate a synthetic a much longer wavelength which extends the ability of the microscope to measure step heights from around 340nm to up to 15 micron.
The unique mapping software allows fast imaging of step heights while maintaining the sub-nanometre resolution achieved with the single wavelength mode.

All DHM configurations provide real-time measurements of topography with sub-nanometer resolution within 200um vertical range.

Reflection DHM is available in three configurations

1.single laser source
2.dual laser sources: short synthetic wavelength
3.three laser sources: short and large synthetic wavelenght

DHM configured with a single wavelength is the ideal tool for measuring smooth surfaces with small local slopes, topography of samples with steps or discontinuities with height smaller than 300 nm within the 200µm live vertical range.

Measuring simultaneously at two wavelengths generates a synthetic wavelength which extends the ability of the microscope to measure step heights up to 2.4um and up to within the 200 μm vertical range.

Working in dual wavelength mode is identical as in single wavelength. It keeps the same ease-of-use and facilities

Benefits of this ultimate optical profiler

Measure dynamic 3D topography of deformable and samples

We are the only one offering such configuration enabling

Real-time imaging   : Video rate acquisition and fast digital reconstruction allow real-time imaging. Dynamic event viewing and active interaction with the observed phenomena are therefore possible.

Robust & stable :  High acquisition rate (from 30 fps up to 1000fps) the short acquisition time (a few microseconds) makes the method insensitive to external vibrations and avoids the need for a vibration isolation table. Its stability permits prolonged examination sequences.

Contactless :  The technique uses low-power light to illuminate the sample and form the image.
It is a full field acqusition method without X/Y scanning within the field of view


 Factory Calibration :  The vertical  Z axis  is intrinsic to phase measurements: it is given by the wavelength of the laser used. The laser sources  used in conjunction with a interferential filter ensures perfect stability, specified to a precision better than 0.01 deg. A calibration with reference calibrated targets, as made for example for AFM, white light interferometers, and for scanning systems is therefore not necessary.


Instrumentation:

Digital Holographic Microscopy (DHM®) is a patented technology exploiting the strength of holography. The system combines DHM with hardware, dedicated software, and specific options to respond to the needs from innovative R&D to industrial applications.

The product range includes microscopes to look through samples (Transmission DHM) and microscopes to measure the reflected wavefront from the sample (Reflection DHM). The DHM technology and products have been developed by Lyncée Tec who also has the competence and flexibility to offer customization and OEM systems.

Roughness Surface roughness is calculated in less than a second
Ra = 37.7 nm Rt = 309.2 nm Rq = 51.8 nm

Ra = 29.1 nm Rt = 207.2 nm Rq = 42.3 nm

Step Height

Surface cracks

Surface cracks reduces the efficiency of solar cells quite a bit since transport of electrons are disturbed.

Fig 1: Control well

Fig 2: HeLa cells treated with Doxorubicin

DHM technology enables to see the livecell  movements online at the same time height related information is obtained due   to DHM interferometry.

Advantages

There is no need of using any contrasting agent,fluorescent tag etc which usually creates some interference in what you want to observe.You will find data acquisition to be very fast & technique is very insensitive to vibrations. It is easy to use.

Applications

It is very useful for  your various biological applicatons.You can keep your cells in various medium, see its response & behaviour in various medium.You can also see effect of toxicity on cells,along with information on cell topography &  height measurements of cells. You can also view effect of drug on cells. You can go through the following link to watch some.

Real time video 16 images/sec in AVI format possible,300fps with post contruction mode also possible.

Z resolution 1nm(microscope objective dependent)

Live quantification of cytotoxicity with DHM. HeLa cells treated with doxorubicin imaged continuously for 24 hours.

Live cell imaging, quantification can be performed without labeling the cells.   https://www.lynceetec.com/biological-imaging-introduction-phase-signal-interpretation/ 

Application page with videos

 1) This application is an assessment of the High Content Screening solution applied to cytotoxicity assays using living cells. https://www.lynceetec.com/cell-based-assays/#tab-1     2) 4D cell imaging allows to study dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. It demonstrates the applicability of DHM® as a tool for label-free quantitative analysis of the cell cycle. https://www.lynceetec.com/4d-cell-imaging/  

MEMS/Nanotechnology Application HF Mirro

(Intensity)                                                              (Phase)

	Driving frequency:40 kHz Pulse length:       125 ns Recovery cycle:    0.5%
	Driving frequency:40 kHz Pulse length:       1 µs Recovery cycle:    4%

Membrane Hysteresis

Goals of the measurement

Gyroscope

In – plane & out- of – plane motion
    

In – plane movement                           Out- of- plane movement

Micro-optics

Micro-technology

Nano-technology

Mems-Moems