Flow cytometry is an analytical technique that can rapidly measure the properties of individual cells or particles as they pass through a beam of light, typically a laser. A flow cytometer takes a sample of cells, transitions them into a single stream and uses lasers and/or light sources to excite biomarkers or labels on the cells to count the number of relevant constituents. The properties measured include relative particle size, relative granularity or internal complexity, and relative fluorescence intensity.

The method requires several photonics components, starting with lasers and other light sources with small, tightly focused beams that can illuminate a single size cell or particle (~30 µm diameter). Laser light with specific wavelengths is needed to excite labels or fluorophores on a cell. Dichroic filters with precision coatings are needed for narrow reflection/transmission bands and high optical density for out-of-band rejection. All photonics components must be precisely aligned and controlled with µm spatial resolution.

A flow cytometer (Figure 1) consists of the three major component systems. The first is the fluidics system that transports sample particles to the laser beam in a narrow, single particle wide stream. The second is the optical system composed of lasers or light sources that illuminate the particles in the sample stream as well as optical filters and beamsplitters that direct the post-sample light signals to optical detectors for counting and processing. The third system is the electronics and signal processing equipment that converts the detected optical signals to electronic signals for processing and analysis.

Schematic of a flow cytometry system
Figure 1. Schematic of a flow cytometry system.

Flow Cytometry Systems

Fluidics System

The fluidics system creates a stream of single particles that can be interrogated individually by the instrument's detection system. The sample is focused by the Bernoulli effect (Figure 2), creating a stream of particles in single file using a method called hydrodynamic focusing. Under optimal conditions (laminar flow), there is no mixing of the central fluid stream and the sheath fluid.

Hydrodynamic focusing produces a single stream of particles
Figure 2. Hydrodynamic focusing produces a single stream of particles.

Optics and Detection

Following hydrodynamic focusing, the particle stream passes through one or more focused laser beams and light scattering or fluorescence emission occurs. The optical output is collected as forward or side scattered light (FSC or SSC) by a PMT or photodiode and the optical data is used to characterize the cell properties. FSC data provides an estimate of a particle's size while SSC provides information on the relative internal complexity of a cell. By combining the FSC and SSC information with fluorescence labeling, it is possible to differentiate cell types in a heterogeneous population such as blood.

The detectors in most flow cytometers are usually PMTs. The specificity of detection is controlled by optical filters. There are three major filter types: long pass filters that transmit light above a cutoff wavelength; short pass filters that transmit light below a cutoff wavelength; and band pass filters that transmit light within a narrow band of wavelengths (see Optical Filter Characteristics for details). These are dichroic filters that block light by phased reflection, allowing only certain wavelengths of light to pass through while interfering with other wavelengths (Figure 3).

Dichroic optical filters
Figure 3. Dichroic optical filters.

When placed at an angle to the oncoming light, a dichroic filter acts as a mirror, allowing it to perform two functions: transmitting specific wavelengths in the forward direction and reflecting the remaining light at a 90o angle. This allows the light path to be passed through a series of filters. The precise choice and order of the filters can be arranged so that multiple signals can be detected simultaneously (Figure 4).

Schematic overview of the optical configuration of a typical flow cytometer setup
Figure 4. Schematic overview of the optical configuration of a typical flow cytometer setup.

Signal and Pulse Processing

Every time a particle passes through the interrogation point, a signal pulse is generated in every detector in Figure 4 with the current from each PMT proportional to the intensity of the scatter or fluorescence signal generated by the cell. These pulses can be mapped by plotting signal as a function of time.

Not all signals that are generated correspond to a particle of interest. PMTs are extremely sensitive and detect signals from irrelevant sources such as stray light, dust, very small particles and debris. The number of these irrelevant pulses can be orders-of-magnitude higher than the number of pulses that are generated by particles of interest. It is therefore desirable and necessary to set a threshold below which non-essential data are ignored. This is done by designating a trigger channel, usually a forward scatter detector, and setting a threshold signal intensity in that channel for recording scattering events. Any pulse that fails to exceed the threshold level is ignored in all detectors; any pulse that surpasses the threshold level is fully processed by the electronics.

The electronics process fluorescence signals for display, analysis, and interpretation. The analog current from the PMT is typically digitized and the pulse height, area, and width determined. The height and area, or maximum and integral, respectively, are used to measure signal intensity since their magnitudes are proportional to the number of photons that reach the PMT. The width of the pulse is proportional to the time that the particles spend in the laser and this can be used to distinguish between single particles or closely interacting particles and doublets.

Applications of Flow Cytometry

 

The ability to simultaneously measure multiple parameters on a cell-by-cell basis is the most powerful attribute of analytical flow cytometry, making it suitable for a wide range of applications. Most commonly, it is used to determine the presence of antigens either on the surface or within cells. In addition, flow cytometry may be used for the analysis of DNA or RNA content, and for functional studies on cells. The following broad range of technological activities employ flow cytometry analytical tools:

  • Medicine
    • Hematology
    • Oncology
    • Immunology
  • Genetic testing
  • Biochemistry and molecular biology, e.g., proteomics, glycomics
  • Marine science
  • Biosynthesis
  • Cell health and biology (including stem cells)
  • Screening
  • Cell cycle analysis
  • Bio Process

MKS Solutions and Products for Flow Cytometry

For over 50 years, MKS has provided components and expertise for thousands of systems for optical applications in a variety of markets. We are a long-term partner to several flow cytometry manufacturers and offer a full range of products with guaranteed performance. With deep understanding of the challenges faced in flow cytometry, MKS is able to partner with customers to develop the best solutions.

Challenges and Solutions

Flow cytometry measures cell properties one cell at a time. As such, the technique works with extremely weak optical signals that can be easily overwhelmed by background noise. High signal to noise ratio (SNR) and high temporal resolution in flow cytometry signals are thus critical requirements for successful measurements by this technique. Achieving these signal characteristics places extreme demands on the quality of the optical, electrical, and mechanical components in flow cytometry systems. For example, laser light sources, optomechanical components, and optical components must have exceptional accuracy and stability, while narrowband filters must be spectrally stable and have high optical density (OD) to minimize background interference. It is often problematic to source and assemble compatible optical/optomechanical components into a flow cytometry system with adequate SNR and temporal resolution.

Challenges in Flow Cytometry MKS Solutions
Maximize signal-to-noise ratio (SNR) High optical density (OD) filters
Maintain alignment between optics and fluidics
(including beam-combining optics)
Robust, stable mounts and positioners
Mounts with lockable positions
Low wavefront distortion mounts
Increase the number of parameters that can be measured CW lasers with a selection of different wavelengths
Optical filters with steep transition lines

MKS supplies many of the critical, high precision components needed in a flow cytometry instrument. MKS lasers have exceptional stability over the widest temperature range in the industry and the best beam pointing stability. Fine pitch position adjusters ensure the alignment is dialed in and maintained for the duration of a cytometry experiment. Tuned mass damping of MKS vibration control solutions effectively isolates cytometers from outside vibration, minimizing resonances, while honeycomb design in these solutions minimizes deflection under load. Beam profilers, along with power meters and detectors are used in signal processing. MKS also provides fully integrated flow cytometry systems through the Optical Solutions Business department.

Flow cytometer system
Figure 5. Flow cytometer system.
Example Solution: Top Adjust Industrial Mounts
Challenge Difficult to place and adjust mounts inside the limited space of flow cytometer HVM-1i
Cause Mirror mounts in use employed traditional back-mounted actuators
MKS Solution Changed optical mounts to top adjust industrial mounts
Results • More efficient use of space inside cytometer
• Long-term stability
Example Solution: Low Wavefront Distortion Mounts
Challenge Not all lasers were focusing to the same point (focal shift issues), leading to sub-optimal SNR HVM-S1w
Cause Too much pressure on beam-combining optics when tightening set screws of mounts ➔ wavefront distortion
MKS Solution Changed optical mounts to low wavefront distortion mounts
Results Reduced focal shift ➔ More accurate focusing ➔ Improved signal-to-noise ratio
Example Solution: Stainless Steel Mirror Mounts
Challenge Needed mounts with low thermal drift for long-term stability SS100-F3H
Cause [new product design]
MKS Solution Stainless steel mounts with lockable hex-adjustment screws
Results Long-term stability as a result of
• Low coefficient of thermal expansion
• Adjustments locked into position
Example Solution: Optical Filters
Challenge Additional optics added to an existing system could affect other channels FG-fluo_cst-S
Cause New laser line added to instrument to allow for use with a larger range of dyes
MKS Solution A matched set of custom-sized high transmission, high OD optical filters with steep transitions
Results • High signal-to-noise ratio for new laser line, without affecting other channels
• Existing hardware could be used to test the new filters
Example Solution: Aspheric Condenser Lens
Challenge Too large of a footprint of the instrument OP-aspherelens-S
Cause Too long of an optical path caused by too many lenses used to focus light onto cells
MKS Solution Replaced multiple spherical lenses with a single aspheric lens with antireflection coating
Results • Reduced footprint (reduced optical path)
• Higher transmission (AR coating & fewer lenses) ➔ Improved signal-to-noise ratio
Example Solution: Motorized Mirror Mounts
Challenge Flow control nozzle requirement of <10 µrad pointing accuracy not being met ➔ increased in-person field service products_DSC_4766
Cause Not enough holding force keeping flow control nozzle in place during shipment
MKS Solution Designed a mirror mount with motorized Picomotor™ piezo actuators to hold the flow control nozzle
Results • Pointing accuracy <1 µrad
• Increased holding force ➔ reduced shipment issues ➔ fewer service calls
Example Solution: Motorized Positioners
Challenge Internal beam routing was experiencing beam drifts ➔ increased in-person field service to realign beam routing AG-LS25
Cause Challenging environment led to temperature fluctuations
MKS Solution Motorized, compact linear positioners to realign beam routing optics
Results Technicians could realign beam routing through their PC ➔ fewer service calls
Example Solution: Complete Components Provider
Challenge Manufacture a designed system 9062-COM
Cause Not the expertise of the designer
MKS Solution Provided mirror mounts, mirrors, optical filters and manual positioners
Results Flow cytometer operating to requirements
Example Solution: Stable Mounts and Positioners
Challenge Needed stable alignment in a space-contrained area MFM-050
Cause [new product design]
MKS Solution Stainless steel flexure mounts and stainless steel manual positioners
Results Long-term stability within allowable space

MKS Products for Flow Cytometry

The table below summarizes typical requirements for flow cytometry systems. Contact us to discuss specific needs of the system in your application.

Entry Level Standard Commercially Available State-of-the-Art
# of Light Sources 1 1 to 5 1 to 16
# of Emission Wavelengths 1 1 to 5 4 to 16
# of Components 5 to 20 5 to 100 10 to 250
Alignment Accuracy Microns Nanometers Nanometers
Bandpass Filters Not always needed Required Critical, with steep transitions
Low Wavefront Distortion Mounts Not as important Required Critical

Check out MKS recommended products for flow cytometry below and select products that best fit your system.

Opto-Mechanics

Suprema
Suprema
Suprema_ZeroDrift
Suprema ZeroDrift
INDUSTRIAL_HVM
HVM Top-Adjust
PERFORMA-i
Performa-i
Optic Diameters 0.5, 1 and 2 in. 0.5 and 1 in. 0.5, 1 and 2 in. 0.5, 1 and 2 in.
Resolution 50, 100, 127 and 254 TPI 100 TPI 80 and 100 TPI 80 and 100 TPI
Angular Range ±4° to ±7° ±4° ±2.5°, ±3° and ±3.5° ±3° and ±4°
Material Stainless Steel Stainless Steel Stainless Steel
Anodized Aluminum
Anodized Aluminum
Drive Types Knob
Hex Key
Knob
Hex Key
Hex Key Hex Key
Lockable Versions Yes Yes Yes Yes
Low Wavefront Distortion Versions Yes Yes Yes No
Other Features Clear-Edge Version
Front- and Rear-Loading Versions
Right- and Left-Handed Configurations
Thermal drift compensation
Front- and Rear-Loading Versions
Slim Profile
Alignment Pin Holes
Front- and Rear-Loading Versions
Slim Profile
Threaded Aperture Version for Thin Optic
Right- and Left-Handed Versions
Picomotor™ Piezo
Optic Diameters 1, 2, 3 and 4 in. kinematic-breadboard-adaptors
Angular Range ±3°, ±3.5°, ±4° and ±5°
Resolution 0.7 µrad angular
Material Anodized Aluminum
Drive Types Picomotor Actuator
Lockable No, but no movement when no power is applied
Low Wavefront Distortion Versions Yes (Stability™ series)
Other Features Clear-Edge Version
Right- and Left-Handed Configurations
multi_axis_lens_pos
Precision LP
OM-5AX_2AX-S
Compact LP
LT_Series
Lens Tube
Optic Diameters 0.5, 1 and 2 in. 1 in. 0.5, 1 and 2 in.
Resolution 100 TPI (Z-axis: 80 TPI) 100 TPI (Z-axis of 9841: 80 TPI)
Adjustments XY
XYZ
XYZ θxθy
XY
XYZ
XYZ θxθy
Material Aluminum Aluminum Aluminum
Drive Types Knob w/ Hex Hole & Knurled Ring Knob w/ Hex Hole
(9841: Knob Only & Knurled Ring)
Lockable Versions Yes Yes
Other Features Zero-free play XY mechanism
Adapters for other optics
Adapters for other optics
Optional lock nuts
Many accessories and adapters
Custom Component and OEM Design

If our standard products satisfy most of your requirements but maybe not all of them, we have the ability to customize them for your needs. MKS has designed thousands of opto-mechanical and positioning products for many decades, so we have the expertise to provide the optimal solution for your application.

One type of customization we’ve done over and over is modifying one of our standard products so that it can exactly meet your needs. We also call this a “same-as-except” type of component. Some examples include:

  • Adding mechanical characteristics to meet space constraints or mounting requirements of your system, such as adding alignment pinholes
  • Modifying mounts to hold non-standard sized optics or non-optical components such as flow cells, sensors, or laser diode modules
  • Inserting higher or lower tensions springs
  • Increasing the travel range of a positioner

Another modification we can make to our components is to use materials, lubricants, adhesives and other design features and manufacturing processes so that they can meet challenging and extreme environments, such as vacuum and cleanroom.

custom-component

We can build-to-print from your own design or help you build entirely new products, whichever is the preferred method of a partnership that you choose. In addition to our design expertise, we have extensive testing capabilities to make sure the products will perform, and we have full ISO certification for our world-class operations.

Optics

Filter Type Excitation Dichroic Emission
Passband Transmittance ≥90% ≥90% ≥90% HPF_Filters
Dichroic Reflectance N/A ≥90% N/A
Spectral Blocking ≥OD6 N/A ≥OD6
Size Ø25 mm 25.5 x 36 mm Ø25 mm
Spectral Response Stable performance @ 0% to 100% RH
Coating Operating Temperature -100° C to 300° C
Other Features Coating hardness, abrasion resistance, adhesion, and humidity to MIL standards
Custom Fluorescence Filter

MKS has been manufacturing optical filters for over 50 years. This includes providing unsurpassed fluorescence filter solutions for applications such as flow cytometry, DNA sequencing, in-vivo imaging, and other fluorescence-based instrumentation.

Our filters are manufactured with our Stabilife coating technology. We use a patented process for the deposition of metal oxide thin film coatings which produce highly dense thin film coatings with extraordinary hardness, abrasion resistance and adhesion to the substrate. As a result of our manufacturing processes and experience, we are able to deliver filters with extremely high transmission, steep transitions from transmission to rejection, and superior image quality. Additionally, our filters provide very high spectral stability over temperature and humidity changes.

custom-filter

We can custom design and manufacture filters that meet your application’s requirements. We’ll work with you to understand the kind of performance specs, size and shape you need whether it’s for emission bandpass, excitation bandpass or dichroic filters. And the quantities we can provide are scalable as your needs grow.

  • Aspheric Condenser Lenses: combine the performance of multiple spherical lenses to reduce the optical path length and number of components.
Aspheric Condenser Lenses
Wavelengths MgF2 coated: 400-700 nm
Uncoated: Visible to NIR
OP-aspherelens-S
Avg. Reflectivity per Surface <1.5% MgF2 coated
4% uncoated
Diameters 6.8 to 75 mm
Effective Focal Lengths 5.87 to 49.24 mm
Shapes Plano-Convex
Bi-Convex
Substrate Materials Schott B 270® Ultra-White Glass
Other Features Reduced Spherical Aberration
Molded Fabrication
Wavelengths 250-600 nm 450-700 nm 480-20000 nm 650-20000 nm
Coatings UV Aluminum Aluminum Silver Gold mirrors
CW Damage Threshold 100 W/cm2 100 W/cm2 1000 W/cm2 200 W/cm2
Reflectivity >90% >93% >96% >96%
Diameters 0.5 to 8 in.
Other Shapes Square, Elliptical, D-shaped, Concave
Substrate Materials Borofloat 33, Zerodur
Other Features Insensitive to polarization and AOI

Positioners

906x-ppp
906x Series
MC-AG-LS25_XZ
Agilis™
SAG-LS-Stacked
Super Agilis™
Drive Type Picomotor™ Piezo Motor Piezo Motor
Travel Range 5, 12.7 and 25.4 mm 12 and 27 mm 16, 32 and 48 mm
Minimum Step Size <30 nm 50 and 100 nm 25 nm (closed-loop)
100 nm (open-loop)
Angular Deviation <100 µrad <200 µrad <150 µrad
Speed 0.02 mm/s 0.5 mm/s 10 mm/s
Bearings Ball Ball Crossed-Roller
Material Stainless Steel Stainless Steel Stainless Steel
Other Features Pre-assembled XY and XYZ configurations
Picomotor and Micrometer combined driven versions
Holding force locks position when idle
Closed-loop controller version
Holding force locks position when idle
Includes controller
Ultralign
Ultralign
DS Dovetail
Industrial DS
Travel Range 13 and 25 mm 6.35, 14 and 25.4 mm
Axes of Travel X, Z, XY, XZ and XYZ X, Z, XY and XYZ
Angular Deviation <100 µrad
Bearings Crossed-Roller Dovetail
Material Stainless Steel Aluminum
Drive Type Adjustment Screw
Micrometer
Motorized Actuator
Hex Key
Lockable Yes Yes
Other Features Extra thick plates for more stability
Low profile
Right- and left-handed configurations
LaserClean™ Version
Right- and left-handed configurations

Lasers

  • Excelsior® One™ CW Lasers: largest portfolio of up to 12 different wavelength options with laser head and controller in a single, compact package.
Excelsior® One™ CW Laser
Output Power 10 mW to 500 mW (adjustable) Excelsior_One
Wavelengths 375 to 1064 nm (free space)
Linewidth <0.01 pm to <1.5 nm
Beam Pointing Stability <6 µrad/°C
Power Stability <±2% over 8 hours
Dimensions 100 x 40 x 40 mm
(3.94 x 1.57 x 1.57 in.)
Other Features Fiber-coupled option
Multi-mode and single freq options
RS-232 software interface

Detectors and Power Meters

Spectral Range 400-1100 nm 200-1100 nm
Detector Material Si UV Enhanced Si 918D-ST-SL
Sensor Size 10 x 10 mm
Max Measurable Power
(w/ OD3 attenuator)
2 W 200 mW
Min Measurable Power
(w/o attenuator)
20 pW
Calibration Uncertainty
(w/o attenuator)
±1% to ±4%
Rise Time ≤3 µs
Other Features Switchable OD3 attenuator
DB15 connector
2938-R_front_angle
1938-R Touchscreen Power Meter
1919-R_angle
1919-R Handheld Power Meter
  • Sub-pW noise level
  • Time-stamped 10 kHz data acquisition rate
  • Trigger In and TTL Out for synchronized measurements
  • Graphical, touchscreen interface
  • USB & RS-232 interfaces
  • Portable use
  • For transmission checks "in the field"
  • USB & RS-232 interfaces

MKS Semiconductor Handbook Cover

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