Our phase contrast techniques

Introduction

QUANTITATIVE PHASE IMAGE | Meaningful pixel values


Each specimen introduces a delay in the light path when light propagates through it. This amount of delay (the phase shift or the Optical Path Difference) is measured by Phasics technique. An image is thus obtained for which each pixel value is the measured local phase shift. More precisely the pixel value is related to the physical thickness and the specimen local refractive index.
 
Phase contrast generation:
Phase-shift-image-principle

 

 

For

HIGHLY VALUABLE BIOLOGICAL PARAMETERS | Dry mass, density, morphology…


This pixel value is meaningful in cell biology as it directly relates to the specimen local mass density. The pixel value sum over the cell area is proven to lead to the cell dry mass, i.e. its weight without water.
cell-dry-mass-measurement

Single cell dry mass and morphometry

Dry mass and other phase related parameters (density, peak…) are proven indicators of many cell mechanisms. Combined to standard morphology features such as surface, they can be used as new label-free biomarkers to quantitatively study and monitor many biological events or pathologies:

  • cell viability (apoptosis detection…)
  • cell growth (cell cycle status, , proliferation…)
  • cell differentiation
  • cell polarity
  • cell motility
  • cell abnormalities: shape change, heterogeneity, presence of parasite…

 

 

All these measurements can be made for individual specimens, such as cells, bacteria, cell aggregates, intracellular components… The technique applies to different cell types: isolated or confluent adherent cells, stem cell colonies… Summed over a complete field of view it estimates the biomass.

Advantages

DRY MASS | at single cell level


New label-free biomarker:

  • Cuantify cell events and pathologies: RBC disorder, cancer cell growth…
  • Monitor cell population over time: cell proliferation, stem cell differentiation, biomass production…

 
Quantitative cell monitoring

MORPHOLOGY PARAMETERS
With high accuracy


Artefact-free and low noise images for:

  • Robust segmentation and measurement
  • Live cell tracking and cell motility measurement
  • Intracellular components observation including cell membranes

Label-free-cell-segmentation

HIGH CONTENT IMAGING
Study each single cell of a large population


  • Run quantitative and statistical study of large cell population based on multiple parameter measurement: dry mass, surface, density…
  • Classify cell into subpopulations

 
subpopulation sorting

LABEL-FREE IMAGING
Non-invasive and fast preparation


  • Time lapse imaging over long period of time
  • No photobleaching

 
Intracellular imaging

Introduction

Phasics offers a unique quantitative phase imaging solution for label-free microscopy. It applies to live cell imaging and delivers artifact-free images which enable accurately measuring valuable biological parameters for each single cell: morphology, dry mass…

Set Up

Phasics technique consists in a camera-like instrument. It plugs on to any optical microscope. A dedicated software package computes the camera image in real time to deliver the quantitative phase image. The technique enables label-free live microscopy without any change on the microscope. Thus it provides artifact-free images for accurate measurement. It is also easy to integrate in very various experiments (time-lapse microscopy, high content screening, cell culture monitoring…)

Camera for quantitative phase microscopy
Phasics technique measures the phase shift thanks to a patented technology: the quadriwave lateral shearing interferometry.

Advantages

PLUG & PLAY CAMERA
Any microscope – any modality


Suitable to:

  • Any microscope for versatile use in Research labs and imaging facilities
  • Any dishes, multi-well plates and any cell culture conditions
  • Incubator for long duration time-lapse

Easy set-up

  • Easy to integrate in very various experiments (time-lapse microscopy, high content screening, cell culture monitoring…)
  • Easy multi-modality
  • Possible OEM integration

REAL TIME
1 acquisition = 1 image


  • Applies to fast live cell event
  • No blur due to cell motion

LABEL-FREE
Easy & non-invasive


  • Fast assay preparation: no staining, no labelling
  • Non-invasive monitoring: long period time-lapse microscopy
  • Non-invasive cell classification and selection

ARTIFACT FREE
as the light is not filtered nor modified


  • Robust segmentation to enable automated analysis
  • Accurate measurement: <2nm accuracy
  • Reliable high content study over large population
  • Phase noise < 0.5nm

Introduction

Phasics offers a unique solution for easily combining fluorescence and quantitative phase microscopy. Thanks to a smart add-on, the same camera captures images from both modalities. So the merging is immediate and the set-up is very simple.
Phase and fluorescence microscopy

 

Merging both techniques provides a very comprehensive dataset on the specimen for deep understanding of biological events:

  • Quantitative phase imaging offers great contrast for label-free cell imaging and tracking and allows easily measuring cell morphological parameters and cell dry mass
  • Fluorescence microscopy provides highly specific information related to the presence of fluorophores for identification of targeted components at cellular or molecular levels

For

MOLECULAR & PHYSIOLOGICAL DATA
At single cell level


  • Correlate molecular pathways and cellular data to understand complex biological event

 

VALIDATE PHASE MEASUREMENT
Use phase to identify elements


  • Use fluorescence to calibrate phase information
READ MORE

ASSIST FLUORESCENCE IMAGING
Use phase to see and track cells


  • See the environment of the labelled specimens
  • Check fluorescence expression
  • Prevent useless photo-bleaching by targeting cells of interest through artefact-free phase imaging

Set Up

Quantitative phase and fluorescence microscopy merging is easy and fast with Phasics solution. The same camera acquires images from both modalities thanks to a smart add-on, the SID4-Element. This add-on integrates Phasics patented technology. With this solution, merging is immediate and set-up is very simple.
Phasics patented technology stands out for its integration as a simple camera at the end of the microscope. Thus it easily combines to other modalities such as fluorescence microscopy. With SID4 Element add-on, switching between fluorescence and phase only requires a deflector
 
Note: this set-up can also apply to other multi-modality experiment

Phase and fluorescence microsocopy-set-up

Advantages

IMMEDIATE MERGING
by using the same camera


  • Quantitative phase image and fluorescence images are acquired with the same camera so they immediatly overlay

SIMPLE SET-UP
No change on the microscope


  • No impact of phase set-up on fluorescence data
  • Artefact-free phase and fluroescence images
  • Any plate are compatible, including multiwell plates

SIMULTANEOUS INFORMATION
at both cellular and molecular level


  • Switching between modalities is immediate

MULTIMODALITY TIME-LAPSE
Elaborated acquisition schemes


  • Acquisition of Phase and fluorescence images can be planned so as to use phase to track cell and use fluorescence only when needed

Introduction

Phasics offers an easy and accurate solution for birefringence imaging. It provides specific contrast enhancement for structured specimens such as collagen in tissues or stress fibers in cells. This is of huge interest for:

  • Digital pathology
  • Fiber detection and identification in air pollution (asbestos…)

Set Up

Phasics set-up for birefringence imaging is very immediate. It relies on a single rotating polarizing element placed at the light source level and Phasics camera-like instrument for QPM. By measuring phase shifts for different light polarization directions, Phasics technique obtains local birefringence data. A “retardance” image is obtained for which each pixel is the local value of linear birefringence introduced by the specimen. In this image, all birefringent structures such as fiber appear with high contrast and can be identified thanks to their pixel values. The slow optical axis along the fiber is also locally identified.

Polarization microsocpy

Advantages

SPECIFIC CONTRAST FOR FIBERS
With birefringence imaging


  • Quantitative data for structure identification on the image
  • Collagen network architecture in tumorous tissue

collagen without labelling

LABEL-FREE FIBER IMAGING
With birefringence imaging


  • Fast protocol
  • Label-free identification of actinQuantitative data for structure identification on the image

SIMPLE SET-UP
With only one polarizer


  • Simple set-up with a single additional element
  • Suitable to any microscope

DIRECT MERGING WITH QPM
With the same camera


  • A QPM image can be acquired with the same camera by just removing the polarizer
  • A merged image on which cells and fibers are contrasted is obtained

Introduction

Phasics offers an innovative solution for quantitative phase tomography. It is perfectly suited to thick tissue imaging: it offers highly contrasted images in the tissue depth without label.

Set Up

The solution relies on the axial scanning of the specimen. For each position data are acquired with Phasics camera-like instrument. They are computed to build the quantitative phase tomographic image.

Introduction

MEASUREMENTS IN THICK TISSUE
Accurate quantitative data


  • Valuable quantitative parameter measurements thanks to an unique technique for quantitative phase tomography

Easy & robust set-up
Simple axial scanning


  • ease of use
  • easy switching between classical QPM and tomography