Tuesday, April 28, 2015

Lab Series #3: Flow Cytometry

Greetings

One technique that has been under constant modification and up gradation, is the cell analysers. From the time Rayleigh (1880) has come up with stability of fluidics jet systems, improvements in cell counting and analysis has come a long way. Today most laboratories (Especially research) is exclusively dependent on Flow cytometers or their improved versions for most of the cell work ups. From about 1980's, Becton Dickinson's flow cytometers have become common cell analyzers in hematology laboratory. 

Photo 1: Leonard Arthur Herzenberg.
Source
With that note, let me talk about flow cytometry. From the initial, classic flow cytometer with a single laser beam, with 2 detectors (one forward one side scatter), the modern day FACS (Fluorescence Activated Cell Sorting) machines have evolved to multi color analyzers, analysing more than 12 colors with multiple detectors (These are referred as Hi-D FACS). Thats a remarkable improvement. Herzenberg's group at Stanford University was the first to design and patent a FACS. Later in 1974, Becton Dickinson, licensed the technology and introduced the first commercial flow cytometer, which was called the FACS-1. But the real successful implication of flow cytometry method, probably came with the development of high-speed flow cytometer by Joe Gray's team that eventually came to be used for sorting human chromosomes in the Human Genome Project. The modern day "Hi-speed flow cytometer" can enumerate and distinguish cells in a mixed population at a rate of 500 to 5,000 cells per second. 

Fig 1: Flow Cytometer components. Source
So whats the basic mechansim of working of a flow cytometer? The simplest way I can put it is analysis of cell by measuring its imparted chromic properties (one at a time), in a constant flow. Let me elaborate. The whole setup consists of follwoing key components- Laser, fluidics, optics and electronics.

Laser stands for "Light Amplification by Stimulated Emission of Radiation". Lasers have the advantage of precision cause they have a very small scatter at great lengths. There are several types of lasers used in flow cytometers. The most common lasers preferred for biophotonic analysis are Argon gas lasers, Green and Yellow diode-pumped solid-state (DPSS) lasers, femtosecond fiber lasers, Supercontinuum lasers etc. DPSS lasers come in several set of varieties. Of the varieties, Nd:YAG (Neodymium doped Yttrium Aluminum Garnet), Nd:YLF (Neodymium yttrium lithium fluoride) and Nd:YVO4 (Neodymium doped Yttrium ortho-Vanadate) laser can cover a range of fluorescence excitation wavelengths. 

The second important component is the Fluidics. The important part of analysis is analyzing each cell one at a time. That means clustered cells cannot be analyzed. This requires that the cell moves in a single line one after the other. This achieved through fluidic design. The fluidics is supplied by a reservoir of liquid, called sheath fluid (pressurized with room air) and taken towards the illumination point. This is called as flow cell/ chamber. The flow cell chamber on the outside is usually made of quartz.

The sheath fluid should not interfere with cell integrity, for this purpose buffers are used. For most mammalian cell lines, phosphate-buffered saline solution is used as sheath fluid. Since the sheath fluid in itself is at high pressure, the sample to be analyzed is injected into the sheath fluid at a higher pressure. This leads to a formation with core made up of cells moving linearly in a single line and the outer sheath fluid. The amount of pressure can be controlled, thereby changing the width of the core allowing size exclusion or inclusion. For example, increasing the sample pressure increases the flow rate by increasing the width of the sample core stream. Such a type of fluidic flow with core and sheath based on pressure is called as coaxial flow and effect is as hydrodynamic focusing.

Fig 2: Scatter of light. Source
So now you have a laser that throws the light you want. The fluidic system brings the cells in a single pile. The light hits the cell. This leads to light scatter of two types- Forward and Side scatter. This is where the optics steps in. The light that is scattered, is detected by using detectors, one in line with the laser (to detect forward scatter, deviating up to 20 degree angle) and one perpendicular to the laser (to detect side scatter at an angle of 90 degree). A bar (Barrier filter) is also placed in front of the forward scatter channel (FSC) and Side scatter channel (SSC), to remove the effect of unsacttered light entering directly.

By using a variety of dichroic mirrors and optical allignment multiple different wavelengths can be detected from one single signal originating from the cell analyzed for. The final detectors are basicaly photomultiplier tubes (PMT) that can be detect specific data.

Fig 3: Filters and Detection paths. Source
In this case (Fig 3) the side scatter containing multiple wavelengths pass through filter 1, filtering the blue light and focusing it to 1st detetctor SS blue. The rest of the wavelength passes through and green is detected by same mechanism in 2nd detector. The last remaining wavelength set is filtered at 3rd dichroic mirror and detected for orange. The final remaining wavelength is directly detected at PMT-red. By adding multiple such "scavenge and focus" detection method, we can detect multiple signals at any given point of time. The practical technical details is not as simple as shown and often requires tricks in optics to get it right.

So far so good. But how did you get the cell to emit the signal at the first place? Simple. You can specifically color different cells with specific colors using immunofluorescence. So if you want to count say CD4 T cells, tag an antibody against CD4 that has a fluorochrome attached. Based on scatter plot generated using side scatter (number of signals) the number of cells can be counted.

Fig 4: Forward scatter for
determining size.
If you have noted the side scatter was used to find the color and count. So what's the importance of forward scatter? As I mentioned already forward scatter can detect usually a scatter of light at an angle of maximum 20 degrees. Hence it is sometimes also called as "Low-angle forward scatter". The the scatter will depend on the size of the cell. Look at the Fig 4 for illustration.

In a nutshell, the flow cytometer, gathers the cell you want to count and analyze in single file, with help of laser finds the right number and other data regarding the cell, transforms the data into a scatter plot. But there is more. We can go a step further. If you need to recover a specific set of cells for further analysis then you can do so by cell sorting. This can be done by a variety of methods such as Electrostatic Cell Sorting (Link).

Further Reading:

1. Herzenberg etal. The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Clinical Chemistry October 2002 vol. 48 no. 10 1819-1827. Link

2. M Nunez Portela et al. A single-frequency, diode-pumped Nd:YLF laser at 657 nm: a frequency and intensity noise comparison with an extended cavity diode laser. 2013 Laser Phys. 23 025801. Link

3. Tkaczyk ER, Tkaczyk AH. Multiphoton flow cytometry strategies and applications. Cytometry A. 2011 Oct;79(10):775-88. Link

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