Flow Cytometry Overview

Flow Cytometry

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.

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.

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).

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).

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.

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
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
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
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
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
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
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
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
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
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-Mech

Optics

Positioners

Lasers

Light Analysis

Opto-Mechanics

Suprema® Stainless Steel Mirror Mounts: the industry's best thermal performance for long-term stability.
Suprema® ZeroDrift™ Mirror Mounts: 85% improved optical pointing stability through thermal drift compensation.
HVM Top-Adjust Industrial Mirror Mounts: for use in compact spaces so your hands do not have to cross the beam path for adjustment.
Performa-i Mirror Mounts: economic, industrial mirror mounts optimized for OEM applications.

	Suprema	Suprema ZeroDrift	HVM Top-Adjust	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 Mirror Mounts: automated, ultra-fine resolution adjustments.

	Picomotor™ Piezo
Optic Diameters	1, 2, 3 and 4 in.
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

LP Precision Multi-Axis Lens Positioners: highest performing lens positioners.
Compact Lens Positioners: ideal solution for applications with limited space.
LT Lens Tubes: combine several optical components into stable, rigid assemblies.

	Precision LP	Compact LP	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.

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.