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LIFE SCIENCES AND MEDICAL DEVICES

FLOW CYTOMETRY
 

Optics Enabling
Flow Cytometry

Flow Cytometry is an analytical technique used in a variety of life science applications for counting, inspecting or sorting particles in solution such as single cells. This technique enables the analysis of mixed cell populations - for example from blood, bone marrow, or even from solid tissues such as tumours when these are dissociated into single cells. This allows for the quick and accurate diagnosis of a variety of diseases or sorting of cells for further analysis. Flow cytometry is used in a variety of disciplines such as immunology, cancer, virology and molecular biology, as well as infectious disease monitoring.

Flow cytometers are diagnostic devices that use optofluidic systems in which one or multiple lasers are focused onto the sample being analysed as particles flow past. This produces either scattered or fluorescent light signals depending on the particle characteristics: particle shape, size or dye used for staining. These signals then pass through filters to the detector(s) - typically photodiodes or photomultiplier tubes. The beam path of these systems usually consists of lenses, optical filters, mirrors, prisms and other optical components to direct the light. The optical components involved are crucial in improving the accuracy of these systems and successfully delivering the signal from the flow cell to the detector(s).

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Blood and Optics

Flow Cytometry Overview & Typical Beampath

Flow cytometry is the primary technology for inspection and detection of blood and other bodily fluid ailments. A flow cytometer is made up of three critical sub systems – a fluidics system, an electronic detection system, and an optical system.

Flow Cytometry
Figure 1: A typical flow cytometer setup

Fluidic System: Hydrodynamic Focusing

High flow rates used for qualitative measurements:

  • Phenotyping, or predicting an organism's phenotype using only genetic information gathered from DNA sequencing or genotyping

Lower flow rates used for higher resolution:

  • Cellular and DNA analysis

Electronic Detection System

Forward-scattered light: measurement of diffracted light slightly off-axis of the laser beam, which detects particles within a given size range

Side-scattered light: measurement of mostly refracted and reflected light at any interface in cell where refractive index changes that is proportional to cell complexity and granularity

Equipment

  • Photomultiplier tubes (PMTs): used to detect the weak signals generated by SSC and fluorescence
  • Photodiodes: less sensitive than PMTs and used to detect stronger FSC signals

Optical System

Excitation Optics: laser and lenses for shaping and focusing of laser beam

Emission Optics: various lenses to collect scatter and mirrors, filters, and beamsplitters for proper routing

Fluorophores and Optical Filters for Fluorescence Microscopy

Learn more about the how fluorophores and optical filters work for flourecsence microsocpy.

Many techniques and methods are used in order to view, diagnose, and treat blood and other bodily fluids. The most common techniques include Flow Cytometry, Cell Sorting, Optofluidics, and Microscopy.

Cell Sorting

Fluorescence activated cell sorting (FACS) is a specific branch of flow cytometry that actively sorts a heterogeneous collection of cells into various containers a single cell at a time. This is done using general light scattering and fluorescence principles based off of each cell’s characteristics.

Optofluidics

Technology that combines the field of microfluidics with optics. The primary applications include broad covering liquid displays, energy, and optical lenses, but the primary startup company drive is focusing on lab-on-chip devices, biosensors, and molecular imaging systems.

High Throughput Screening

A powerful drug discovery process used heavily in pharmaceuticals. Typically an automated procedure that allows for quicker deployment of novel drugs with less risk for human error.

Microscopy

Traditional light microscopes are used to view histology slides or prepared cells and samples. Higher end microscopes known as confocal or multiphoton microscopes utilize multiple lasers, scanning mirrors, motorized actuators, and an array of high end detectors to better understand intracellular activity or protein-protein interactions.

Fluorescence-Activated Cell Sorting (FACS)

A specialized type of flow cytometry that uses the fluorescent and scattering characteristics of biological cells to sort them into separate containers. It is used for separating in a heterogeneous mixture one at a time.

Diagnostic Techniques

Product Spotlight

Optics are crucial to many life sciences applications and medical devices including flow cytometers. Beamsplitters and various types of filters, such as bandpass, dichroic, longpass, and shortpass filters, are only a few of the most prominently utilized.

Bandpass Filters

Bandpass Filters

Optical bandpass filters are used to transmit a desired portion of the spectrum while rejecting all other wavelengths outside of the pass band.

Figure 2 (left): Transmission profile of a bandpass filter

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Figure 3: A shortpass filter and a longpass filter can be combined to create a custom bandpass filter
Shortpass Filters

Shortpass Filters

Optical shortpass edge filters are used to transmit wavelengths shorter than a specific cut-off wavelength.

Figure 4 (left): Transmission profile of a shortpass filter

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Longpass Filters

Longpass Filters

Optical longpass edge filters are designed to transmit wavelengths greater than the specific cut-on wavelength of the filter.

Figure 5 (left): Transmission profile of a longpass filter

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Dichroic Filters

Dichroic Filters

Dichroic filters reflect unwanted wavelengths while transmitting the desired portion of the spectrum. This effect is desired for some applications because light can be separated by wavelength into two paths.

Figure 6 (right): Transmission profile of a dichroic filter

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Beamsplitters

Beamsplitters

Beamsplitters are optical components used to split incident light into two separate beam paths. Unlike dichroic filters, the input light isn’t separated by wavelength; it is separated into two paths by a defined reflection/transmission ratio such as 50/50 or 70/30.

Figure 7 (left): Incident light is split into two separate beam paths by this plate beamsplitter

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Ailments of Blood

Below are common ailments of the blood that are detected by advanced diagnostic techniques such as flow cytometry. Optical advancements allow these ailments to be more easily detected and treated, providing a means for making medical technology and equipment more quick, portable, and simple to use.

Figure 8: Common ailments of the blood
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